Title: ANALOGUES OF AN ANTI-THROMBIN POLYPEPTIDE AND PROCESS FOR THEIR PREPARATION

The present invention relates to analogues of anti- thrombin polypeptides and to a process for their preparation. The analogues have been obtained by C-terminal modification of anti-thrombin polypeptides from the leech Hirudinaria manillensis and have an improved biological activity in comparison to the polypeptides from which they are derived.

The most popular anticoagulant peptides are probably those belonging to the family of hirudins. Hirudin, originally isolated from the medicinal leech, Hirudo medicinalis. is a well known and well characterized polypeptidic inhibitor of thrombin ¹ - 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 efficacy in preventing venous thrombosis, vascular shunt occlusion and thro bin-induced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity, little or no antigenicity and a very short clearance time from circulation. ³

Polypeptides with anticoagulant properties have been isolated from a different leech species, Hirudinaria manillensis (EP-A-0347376 and WO 90/05143) . This leech is evolutionarily more advanced than Hirudo medicinalis and could therefore synthesize anticoagulant peptides whose amino acid sequences may be different from those of hirudin and other known hirudin variants.

We have analysed a preparation obtained from Hirudinaria manillensis leeches. We have found three polypeptides having anti-thrombin activity, characterized by the following amino acid sequences (European Patent Application No. 92301721.4) PI (SEQ ID NO: 1)

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu 1 5 10 15

Cys Val Gly Gly Asn Leu Cys Gly Gly Gly Lys His Cys Glu Met

20 25 30

Asp Gly Ser Gly Asn Lys Cys Val Asp Gly Glu Gly Thr Pro Lys 35 40 45

Pro Lys Ser Gin Thr Glu Gly Asp Phe Glu Glu lie Pro Asp Glu

50 55 60

Asp lie Leu Asn; 64 P2 (SEQ ID NO: 2)

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu 1 5 10 15

Cys Val Gly Ser Asn Val Cys Gly Glu Gly Lys Asn Cys Gin Leu

20 25 30 Ser Ser Ser Gly Asn Gin Cys Val His Gly Glu Gly Thr Pro Lys

35 40 45

Pro Lys Ser Gin Thr Glu Gly Asp Phe Glu Glu lie Pro Asp Glu

50 55 60

Asp lie Leu Asn; or 64

P3 (SEQ ID NO: 3)

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu 1 5 10 15

Cys Val Gly Ser Asn Val Cys Gly Glu Gly Lys Asn Cys Gin Leu 20 25 30

Ser Ser Ser Gly Asn Gin Cys Val His Gly Glu Gly;

35 40

Amino acid residues are presented according to the three letter code (Eur. J. Bioche . 138. 9-37, 1984) . The invention provides a p ^* olypeptide comprising a sequence represented by formula (I)

P,. ₆₃ -Xaa-Z (I) wherein P, _.ω is the amino acid sequence from position 1 to position 63 of the polypeptide PI (SEQ ID NO: 1) or P2 (SEQ ID NO: 2) , Xaa is any amino acid residue and Z is -OH, -NH ₂ , -Gly-OH or a dipeptide consisting of:

a glycine or a polar amino acid residue, followed by an amino acid selected from the group consisting of: alanine, glycine, valine, leucine, isoleucine, proline, cysteine, methionine, serine, threonine, lysine, arginine, histidine, asparagine, glutamine, aspartic acid and gluta ic acid, with the proviso that when Z is -OH, -NH ₂ or -Gly-OH, Xaa can not be asparagine and a pharmaceutically acceptable salt thereof. The pharmaceutically acceptable salts of the polypeptides may be acid addition salts. They may be salts with an inorganic acid such as a hydrohalic acid, such as hydrochloric acid; sulphuric acid; phosphoric acid; or pyrophosphoric acid. The salts may be salts with an organic acid such as benzenesulphonic, p-toluenesulphonic, methanesulphonic, acetic, lactic, palmitic, stearic, malic, tartaric, ascorbic or citric acid. The analogues also contain free carboxyl groups and may therefore be present as sodium, calcium, potassium, magnesium or ammonium salts or salts with a physiologically tolerable organic nitrogen-containing base. The analogues can also be in the form of inner salts.

The polypeptides of the invention are able to inhibit the proteolytic effects of thrombin, for example fibrin formation from fibrinogen and the activation of factor V and factor VIII. The polypeptides are therefore useful, for example, as anticoagulant and antithrombotic drugs, for the treatment of thromboembolic pathologies such as disseminate intravascular coagulation, cerebral embolisms, deep venous thrombosis and arterial thrombosis. They are also useful in prophylactic treatment of coagulation disorders, for example for the therapy of acute myocardial infarction.

The polypeptides of the invention consist essentially of the sequence P, _.M -Xaa-Z. However, the polypeptides may be preceded by all or part, typically a C-terminal part, of a leader sequence. The leader sequence may be a native leader sequence of a natural polypeptide from which the polypeptides

of the invention are derived, or may be a foreign leader sequence. The leader sequence may be capable of directing secretion of the polypeptides of the invention. Two of the natural anti-thrombin polypeptides are expressed with a leader sequence which is cleaved subsequently. All or part of this leader sequence may therefore . be present in the polypeptide of the invention, the sequence being: Met Phe Ser Leu Lys Leu Phe Val Val Phe Leu Ala Val Cys lie Cys Val Ser Gin Ala (SEQ ID NO: 32) .

A polypeptide according to the invention, or a salt thereof, may be prepared by: 1) isolating an anti-thrombin polypeptide from the tissue or secretions of a leech of the species Hirudinaria manillensis and 2) subsequently modifying the C-terminal end of such a polypeptide. More specifically, the polypeptide can be obtained by obtaining a preparation of leech extract, subjecting the preparation to high pressure liquid chromatography, and modifying the C-terminal end of the polypeptide.

A polypeptide according to the invention or a salt thereof can then be prepared by:

(a) providing a host, transformed with an expression vector comprising a DNA sequence encoding a said polypeptide, under such conditions that the said polypeptide is expressed;

(b) isolating the said polypeptide thus obtaining a polypeptide of the invention wherein X is -OH or Gly-OH or a pharmaceutically acceptable salt thereof, and if desired,

(c) converting the polypeptide in which Z is -OH or Gly- OH into the corresponding C-terminal amidated derivative by way of an amidating enzyme, so obtaining a polypeptide of the invention wherein Z is -NH ₂ or a pharmaceutically acceptable salt thereof.

This approach is typically based on obtaining a nucleotide sequence encoding the polypeptide it is wished to express and expressing the polypeptide in a recombinant

organism. The cultivation of the genetically modified organism leads to the production of the desired product displaying biological activity. The present invention therefore further provides

- an expression vector comprising a DNA sequence encoding a polypeptide of the invention;

- a host transformed with a compatible expression vector according to the invention; and - a DNA sequence encoding a polypeptide according to the invention.

A host in which a polypeptide according to the invention is able to be expressed is prepared by transforming a host with a compatible expression vector of the invention. The expression vector can be prepared by:

(a) chemically synthesising a DNA sequence encoding a polypeptide of the invention; and

(b) inserting the said DNA into an expression vector. Alternatively, an expression vector can be prepared by: (a) producing and isolating a cDNA encoding the polypeptide PI or P2 from mRNA of a leech of the species Hirudinaria manillensis;

(b) utagenising the isolated cDNA so as to obtain a nucleotide sequence encoding a polypeptide of the invention; and

(c) inserting the said nucleotide sequence into an expression vector.

A polypeptide according to the invention is consequently prepared by providing a transformed host under such conditions that the polypeptide is expressed. When a eucaryotic host is employed, the polypeptide can be obtained glycosylated. The polypeptide can be isolated as such or in the form of a pharmaceutically acceptable salt. In this way, a polypeptide or salt according to the invention may be obtained in essentially pure form.

The polypeptides of the invention may be modified by way

of amino acid extension at either or each end. A polypeptide composed of such an extended sequence must of course still exhibit anti-thrombin activity. For example, a short sequence of up to 30 amino acid residues may be provided at either or each terminus.

The polypeptides of the invention may be subjected to one or more post-translational modification such as sulphation, COOH- amidation, acylation or chemical alteration of the polypeptide chain. For example a polypeptide having a glycine residue at its carboxy terminus may be subjected to enzymatic amidation with peptidyl-glycine α-amidating monooxygenase (PAM enzyme) . PAM enzyme catalyzes the C- terminal oxydative cleavage of glycine-extended peptides to give the corresponding amidated product and glyoxylate ⁴ .

The polypeptide P, _.63 -Val-NH ₂ constitutes a particular embodiment of the present invention. P[^ ₃ -Val-NH ₂ is the polypeptide of formula (I) wherein Xaa is Val and Z is -NH ₂ . The polypeptide may be obtained by amidating the polypeptide of formula P^-Val-Gly-OH.

The C-terminal -amide structure contributes to the biological stability of the amidated polypeptide conferring protection from the carboxypeptidase-mediated degradation, increases its in vivo half-life and confers a more selective receptor binding ⁵ . For these reasons, α-amidation represents an important and characteristic post-translational processing of many secreted peptides in eukaryotic organisms. The amidating activity of PAM enzyme has been shown to be dependent upon the presence of molecular oxygen and copper ions and to be stimulated by cofactors such as ascorbate, catalase or potassium iodide ⁶ .

As an alternative to the use of PAM enzyme, other amidating enzymes may be used. For example, carboxypeptidase Y and, more generally the class of carboxypeptidases has been proved to be an efficient amidating enzyme ⁷ . Contrary to the PAM enzyme, carboxypeptidases are able to amidate polypeptides

having any amino acid residue at their C-terminal end.

In order to produce a polypeptide of the invention by recombinant DNA technology, a gene encoding a said polypeptide is prepared. The invention includes DNA sequences consisting essentially of the following sequences:

GTT TCT TAC ACC GAC TGC ACC GAA TCT GGC CAG AAC TAC TGC CTG TGC GTT GGT TCT AAC GTT TGC GGT GAA GGT AAA AAC TGC CAG CTG TCT TCT TCT GGT AAC CAG TGC GTT CAC GGT GAA GGT ACC CCG AAA CCG AAA TCT CAG ACT GAA GGT GAC TTC GAA GAA ATT CCG GAC GAA GAC ATC CTG GTT GGT TAG (SEQ ID NO: 16);

GTT TCT TAC ACC GAC TGC ACC GAA TCT GGC CAG AAC TAC TGC CTG TGC GTT GGT GGT AAC CTG TGC GGT GGT GGT AAA CAC TGC GAA ATG GAT GGT TCT GGT AAC AAA TGC GTT GAT GGT GAA GGT ACC CCG AAA GCG AAA TCT GAG ACT GAA GGT GAC TTC GAA GAA ATT CCG GAC GAA GAC ATC CTG' GTT GGT TAG (SEQ ID NO: 17);

GTT TCT TAC ACC GAC TGC ACC GAA TCT GGC CAG AAC TAC TGC CTG TGC GTT GGT TCT AAC GTT TGC GGT GAA GGT AAA AAC TGC CAG CTG TCT TCT TCT GGT AAC CAG TGC GTT CAC GGT GAA GGT ACC CCG AAA CCG AAA TCT CAG ACT GAA GGT GAC TTC GAA GAA ATT CCG GAC GAA GAC ATC CTG AAC GGT GCT TAG TAA (SEQ ID NO: 33).

The DNA coding sequence typically does not include introns. The DNA sequence is isolated and purified. The gene is inserted in an expression vector able to drive production of the recombinant product. The DNA sequence may be a mutagenized cDNA sequence. The DNA sequence may be a synthetic DNA sequence. 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 six chemically synthesised oligonucleotides, each oligonucleotide

representing about one third of one strand of a double- stranded DNA gene. The oligonucleotides are ligated and annealed to obtain the desired gene. Typically, a gene is constructed with restriction sites at each end to facilitate its subsequent manipulation.

A DNA sequence may be provided which further encodes a leader peptide as mentioned above. The leader peptide is capable of directing secretion of the polypeptide from cells in which the polypeptide is to be expressed. The sequence encoding the leader peptide is typically fused to the 5'-end of the DNA sequence encoding the polypeptide.

The leader peptide may be the OmpA leader peptide when expression in a bacterial host, such as E. coli is required. The leader peptide may be the leader peptide of vesicular stomatitis virus G protein (VSV G protein) when expression is to be in insect cells. Appropriate DNA sequences encoding the OmpA and VSV G protein leader sequences "are: OmpA leader: ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT 42 TTC GCT ACC GTA GCG CAG GCC (SEQ ID NO: 4) 63

VSV G protein leader:

ATG AAG TGC CTT TTG TAC TTA GCC TTT TTA TTC ATT GGG GTG 42 AAT TGC (SEQ ID NO: 5) 48 A DNA sequence may be provided which encodes a fusion protein which is cleavable to release a polypeptide of the invention. A DNA sequence may be used which encodes a carrier polypeptide sequence fused via a cleavable linkage to the N-terminus of a polypeptide of the invention. The cleavable linkage may be one cleavable by cyanogen bromide.

For expression of the polypeptide, an expression vector is constructed which comprises a DNA sequence encoding the polypeptide and which is capable of expressing the 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 polypeptides 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 the polypeptide. The expression vector may be a plasmid. In that case, preferably a promoter selected from the P _lπ , and P _|PP/ , _ac 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 the polypeptide.

An expression vector capable of expressing the polypeptide may be prepared in any convenient fashion. A DNA fragment encoding the 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 the polypeptide into a baculovirus transfer vector at a restriction site downstream of the polyhedrin promoter; and (ϋ) 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 polypeptide gene 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 the polypeptide of the invention. As indicated above, a

cleavable linkage may be provided at the fusion junction. The insect cells employed in step (ii) are typically Spodoptera fru iperda cells. The wild-type baculovirus is typically Autographa californica nuclearpolyhedrosis virus (AcNPV) .

An expression vector encoding the polypeptide is provided in an appropriate host. Cells are transformed with the gene of the polypeptide. A transformed host is provided under such conditions that the polypeptide is expressed. 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 Spodoptera 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 the polypeptide is produced.

The polypeptide that is expressed may be isolated and purified. A polypeptide having the aminoarid sequence depicted in formula (I) above preceded by a Met residue attributable to a translation start codon can be obtained. Alternatively, as mentioned above, a fusion protein may be obtained comprising the amino acid sequence of formula (I) fused to a carrier sequence. The carrier sequence is tipically fused to the N-terminus of the polypeptide of formula (I) .

Where a suitable linkage is provided in the fusion protein between the amino acid sequence of formula (I) and the carrier sequence, a polypeptide having an amino acid sequence according to formula (I) can be released by cleavage with a

suitable agent.

A polypeptide of the invention or a pharmaceutically acceptable salt thereof can also be prepared by: (a) chemically synthesising the said polypeptide; and

(b) isolating the said polypeptide thus obtained or a pharmaceutically acceptable salt thereof.

The polypeptides can therefore be built up by chemical synthesis from single amino acids and/or preformed peptides of two or more amino acids in the order of the sequence of the desired polypeptide. Solid-phase or solution methods may be employed. The resultant polypeptide may be converted into a pharmaceutically acceptable salt if desired.

In solid-phase synthesis, the amino acid sequence of the desired polypeptide is built up sequentially from the C- terminal amino acid which is bound to an insoluble resin.

When the desired polypeptide has been produced, it is cleaved from the resin. When solution-phase synthesis is employed, the desired polypeptide may again be built up from the C- terminal amino acid. The carboxy group of this acid remains blocked throughout by a suitable protecting group, which is removed at the end of the synthesis.

Whichever technique, solid-phase or solution-phase, is employed each amino acid added to the reaction system typically has a protected amino group and an activated carboxy group. Functional side-chain groups are protected too. After each step in the synthesis, the amino-protecting group is removed. Side-chain functional groups are generally removed at the end of the synthesis. A polypeptide may be converted into a pharmaceutically acceptable salt. It may be converted into an acid addition salt with an organic or inorganic acid.

Suitable acids include acetic, succinic and hydrochloric acid. Alternatively, the polypeptide may be converted into a carboxylic acid salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.

A polypeptide or pharmaceutically acceptable salt thereof may be used in a pharmaceutical composition, 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) . A polypeptide according to the invention is an anti-thrombin polypeptide and is suitable for treatment of thromboembolic events, such as the coagulation of blood, typically in a human patient. A polypeptide according to the present invention displays improved biological characteristics when compared to the corresponding native polypeptide from which it is derived. A polypeptide can therefore be used for the therapy and prophylaxis of thromboses and thromboembolisms, including the prophylaxis of post-operative thromboses, for acute shock therapy (for example for septic or polytraumatic shock) , for the therapy of consumption coagulopathies, in haemodialyses, haemoseparations and in extracorporeal blood circulation. In one embodiment of the invention, the polypeptide or salt thereof can be coadministered with a plasminogen activator, such as tissue plasminogen activator. The dosage depends especially on the specific form of administration and on the purpose of the therapy or prophylaxis. The size of the individual doses nd the administration regime can best be determined by way of an individual judgement of the particular case of illness; the methods of determining relevant blood factors required for this purpose are familiar to the person skilled in the art. Normally, in the case of an injection the therapeutically effective amount of the compounds according to the invention is in a dosage range of from approximately 0.005 to approximately 0.1 mg/kg body weight. A range of from approximately 0.01 to approximately 0.05 mg/kg body weight is preferred. The administration is effected by intravenous, intramuscular or subcutaneous injection. Accordingly, pharmaceutical compositions for

parenteral administration in single dose form contain per dose, depending on the mode of administration, from approximately 0.4 to approximately 7.5 mg of the compound according to the invention. In addition to the active ingredient these pharmaceutical compositions usually also contain a buffer, for example a phosphate buffer, which is intended to keep the pH value between approximately 3.5 and 7, and also sodium chloride, mannitol or sorbitol for adjusting the isotonicity. They may be in freeze-dried or dissolved form, it being possible for solutions advantageously to contain an antibacterially active preservative, for example from 0.2 to 0.3% 4-hydroxybenzoic acid methyl ester or ethyl ester. A composition for topical application can be in the form of an aqueous solution, lotion or gel, an oily solution or suspension or a fat-containing or, especially, emulsified ointment. A composition in the form of an aqueous solution is obtained, for example, by dissolving the active ingredients according to the invention, or a therapeutically acceptable salt thereof, in an aqueous buffer solution of from e.g., pH 4 to pH 6.5 and, if desired, adding a further active ingredient, for example an anti-inflammatory agent,and/or a polymeric binder, for example polyvinylpyrrolidone, and/or a preservative. The concentration of active ingredient is from approximately 0.1 to approximately 1.5mg, preferably from 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a gel.

An oily form of administration for topical application is obtained, for example, by suspending the active ingredient according to the invention, or a therapeutically acceptable salt thereof, in an oil, optionally with the addition of swelling agents, such as aluminium stearate, and/or surfactants (tensides) having an HLB value ("hydrophilic- lipophilic balance") of below 10, such as fatty acid monoesters of polyhydric alcohols, for example glycerin

monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A fat-containing ointment is obtained, for example, by suspending the active ingredient according to the invention, or a salt thereof, in a spreadable fatty base, optionally with the addition of a tenside having an HLB value of below 10.

An emulsified ointment is obtained by triturating an aqueous solution of the active ingredient according to the invention, or a salt thereof, in a soft, spreadable fatty base with the addition of a tenside having an HLB value of below 10. All these forms for topical application can also contain preservatives. The concentration of active ingredient is from approximately 0.1 to approximately 1.5mg, preferably from 0.25 to 1.0 mg, in approximately 10 g of base.

In addition to the compositions described above and pharmaceutical compositions analogous thereto that are intended for direct medicinal use in the body of a human or a mammal, the present invention relates also to pharmaceutical compositions and preparations for medicinal use outside the living body of humans or mammals. Such compositions and preparations are used especially as anticoagulant additives to blood that is being subjected to circulation or treatment outside the body (for example extra corporeal circulation or dialysis in artificial kidneys) , preservation or modification (for example haemoseparation) . Such preparations, such as stock solutions or alternatively preparations in single dose form, are similar in composition to the injection preparations described above; however, the amount or concentration of active ingredient is advantageously based on the volume of blood to be treated or, more precisely, on its thrombin content. In this connection it must be borne in mind that the active ingredient according to the invention (in free form) completely deactivates approximately 5 times the amount by weight of thrombin, are physiologically harmless even in relatively large amounts,

and are eliminated from the circulating blood rapidly even in high concentrations so that there is no risk of overdose, even, for example, during transfusions. Depending on the specific purpose, the suitable dose is from approximately 0.01 to approximately 1.0 mg of the active ingredient/litre of blood, although the upper limit may still be exceeded without risk. The following Examples illustrate the invention. In the accompanying drawings: Figure 1 is a chromatogram showing the results of the HPLC analysis of Example 1. PI to P3 denote the three different peaks obtained from the preparation according to Example 1, FT is for flow through and 4-AB is for 4- aminobenzamidine. Figure 2 shows the elution profiles obtained in Example 2(b) for trypsin-digested PE-P1 (A) and PE-P2 (B) .

Figure 3 shows the nucleotide sequence of the six oligos coding for the analogue P2,. ₆₃ -Val-Gly-OH linked to the OmpA leader peptide. P2,. ₆₃ -Val-Gly-OH is an analogue of Formula (I) wherein P is the anti-thrombin polypeptide P2, Xaa is Val and Z is Gly-OH. The sequence shown in bold face indicates the OmpA leader peptide; the Hind III, Bal I and BamHI restriction sites are underlined and the codons for Val^ and Gly _6S are in italics. Figure 4 shows the scheme of the construction of the plasmid, named 0MP-P2VG, which is the source of a Bal I-BamHI DNA fragment for further genetic constructions.

Figure 5 shows schematically the construction of pFC- P2VG which is the plasmid used for the production of P2,. ₆₃ - Val-Gly-OH protein in E. coli.

Figure 6 shows the general structure of the plasmid pOMPA-P2VG used for the production of P2,. ₆₃ -Val-Gly-OH in E. coli. We employed traditional gene manipulation techniques to prepare this new plasmid l^re the P2,. ₆₃ -Val-Gly-OH gene is under transcriptional control of the hybrid promoter P _lpp/lac . Even in this case, the OmpA leader peptide drives secretion

of P2,. ₆₃ -Val-Gly-OH to the periplasm of E. coli.

Figure 7 shows the nucleotide sequence and assembling of the synthetic oligos used for the secretion of P2,. ₆₃ -Val-Gly- OH from insect cells. The sequence shown in bold face indicates the VSV G protein leader peptide.

Figure 8 is a schematic representation of the construction of a new recombinant M13, named VSV-P2VG, where the complete P2,_ ₆₃ -Val-Gly-OH gene is linked to the VSV G protein leader peptide.

Figure 9 shows schematically the construction of pAc- P2VG which has been used as transfer vector to the baculovirus genome. pAcYMl is the starting plasmid widely used as acceptor of heterologous sequences to be transferred to the virus.

Figure 10 shows the nucleotide sequence and assembling of the synthetic oligos coding for the beginning of the P2,. ₆₃ Val-Gly-OH chain. The ATG codon coding for the additional methionine residue is shown in bold face. Figure 11 shows schematically the construction of pAcFTl, which has been used for intracellular expression. Figure 12 is a schematic representation of a new transfer plasmid, named pAcFTl-P2VG, which carries the complete P2,. ₆₃ - Val-Gly-OH sequence linked to the first 18 amino acids of polyhedrin. This plasmid has been used to transfer the heterologous sequence to the baculovirus genome.

Figure 13 shows the nucleotide sequence of the six oligos coding for the analogue Pl^-Val-Gly-OH linked to the OmpA leader peptide. Pl,. ₆₃ -Val-Gly-OH in an analogue of Formula (I) wherein P is the anti-thrombin polypeptide PI, Xaa is Val and Z is Gly-OH. The sequence shown in bold face indicates the OmpA leader peptide; the Hind III, Bal I and BamHI restriction sites are underlined and the codons for Val _M and Gly _6S are in italics. Example 1

An antithrombin preparation was prepared from

Hirudinaria manillensis leeches according to the procedure illustrated under a) to d) below: a) Acetone extraction Ethanol dried leech heads (2920 g) were finely chopped into small pieces and treated with a mixture 40:60 acetone/water (7.5 1) . After homogenisation with stirring at room temperature, the mixture was spun for 15 min at 2,700 rpm and the supernatant was decanted; the pellet was again resuspended in 40:60 acetone:water mixture, stirred for 30 min and the mixture centrifuged for 15 min at 2,700rpm. The supernatant was pooled with the initial one and acidified to pH 4.5 with glacial acetic acid (vol. 8.5 1) . The mixture was spun at 2,700 rpm for 15 minutes, then the supernatant was decanted and the pH of the solution adjusted to pH 6.0 by adding 30% ammonia. Following rotary evaporation at 35°C, the pH of the concentrated solution was lowered to 1.8; precipitated contaminants were removed by centrifuging and the raw anti- thrombin material was precipitated from the mixture using a 9-fold acetone excess. The mixture was then spun down, the pellet resuspended in acetone and again centrifuged. The precipitated material was then lyophilized. b) Ionic exchange purification The raw anti-thrombin material was reconstituted in water, dialyzed against 10 mM ammonium acetate buffer at pH

4.0 and loaded onto a carboxymethyl Sepharose column

(CMSepharose, Pharmacia, 2.6 x 30 cm) pre-equilibrated in the same buffer. Following a 100 ml washing with starting buffer, anti-thrombin active fractions were eluted with 20mM ammonium acetate pH 4.5, collected and pooled (1.3 1) . For further purification steps, pooled fractions were concentrated to 0.5 1 in a Minitan apparatus (Millipore) ; the concentrated solution was neutralised with NaOH and then applied on to a Q Sepharose column equilibrated in 20 mM Tris-HCl pH 7.0. The bound material was eluted with a linear

gradient of 0 - 1 M NaCl in the starting buffer. The fractions containing with anti-thrombin activity were pooled, concentrated and desalted on a Superdex S-200 column eluted with 20 mM Tris-HCl pH 7.5 at a flow rate of 4ml/min. Active pool from gel filtration was concentrated by Minitan and further purified by weak anion exchange chromatography (DEAE FPLC) . The active material was loaded onto a Protein Pak DEAE-5PW column (Waters) and eluted with a gradient of 0 - 1 M NaCl in 20 mM Tris-HCl pH 6.5, at a flow rate of 1.0 ml/min. Active fractions were pooled, characterized for protein content and activity (specific activity: 800 ATU/mg), and freeze-dried in a Speed Vacconcentrator (Savant) .

The thus obtained partially purified material (specific activity 800 ATU/mg) was then subjected to two additional chro atographic steps, in order to get homogenous polypeptides, as described below under c) and d) :

c) Thrombin-Sepharose Commercial bovine thrombin (Sigma) was further purified according to the procedure described by Lundblad ⁹ and then was attached to activated Sepharose CL 6B (Pharmacia) following manufacturer's instructions. The column (1.7 ml) was equilibrated with 50 mM Tris-HCl pH 8.3 and the freeze- dried material from DEAE-FPLC (reconstituted in buffer) was loaded. The column was subjected to three washings, in starting buffer, then in the same buffer containing 3.0 M NaCl and again with starting buffer (each washing was three times the column volume). Flow rate was 0.3 ml/min. The bound material was eluted with 10 ml of 0.1M 4- aminobenzamidine in 25 mM HC1. The active fractions were pooled and buffer exchanged in 50 mM Tris-HCl pH 8.3 onto a PD-10 column (Pharmacia) .

Unbound material eluted from the column by washing in starting buffer and still containing anti-thrombin activity, was reloaded onto the column until all the activity was bound

and chromatographed.

d) RP-HPLC Material obtained after affinity chromatography was finally purified by reverse phase high performance chromatography (RP-HPLC) on a C4 Vydac column (4.6 x 250 mm.5μ) using 20 mM sodium phosphate pH 7.5 as first eluent and 50% acetonitrile in water as modified. Anti-thrombin polypeptides were eluted with a linear gradient from 5% to 55% of eluent B in 45 minutes, at room temperature with a flow rate of 1.0 ml/min. The resulting chromatogram is shown in Figure 1.

Peaks of protein (detected at 220 nm) were manually collected, concentrated under vacuum and re-chromatographed under the same conditions.

Pure anti-thrombin polypeptides after C4 HPLC were characterized for protein content, amino acid composition, N- terminal sequence, C-terminal end and their activity determined by in vitro assays (ATU/NIH test and "thrombin time" test) . Each of the three peaks of protein has been found to be endowed with anti-thrombin activity.

The complete amino acid sequences of the polypeptides labeled PI and P2 in Figure 1 were determined by N-terminal sequencing of the peptides obtained from tryptic and V8 protease digests. The sequences are reported above together with the partial amino acid sequence of the other polypeptide <P3).

Example 2 - Tryptic digestion and peptide mapping of pyridylethylated (PE) PI and P2

a) Reduction/Alkylation - Active fractions purified by affinity chromatography on Thro bin-Sepharose (Example lc) were pooled and buffer exchanged in 10 M Tris-HCl pH 8.3 onto a PD-10 column. The active pool (about 50 μg) was

concentrated in a Speed-Vac centrifuge (Savant) and treated with 100 μl of 1% b-mercaptoethanol in 6M guanidine-HCl / 50 M Tris-HCl pH 8.5, under nitrogen, in the dark, for 2 hours at room temperature. Then 4 μl of 4-vinyl-pyridine (neat) were added and the mixture incubated again for 2 hours as above ¹⁰ .

Pyridylethylated polypeptides were first recovered from the reaction mixture by RP-HPLC on a C4 Vydac (4.6x250mm, 5μm) column eluted with a 90 min. linear gradient from 5-65% acetonitrile in 0.1% TFA, at a flow rate of l.Oml/min. Under such conditions the mixture of anti-thrombin polypeptides is poorly resolved so that they have to be re-chromatographed on the same column using the elution system sodium phosphate/acetonitrile with the conditions already described in Example Id. b) Trypsin digestion and peptide mapping of PE-P1 and PE-P2 - Purified PE-P1 and PE-P2 (respectively 10 and 20 μg) were digested with TPCK-treated trypsin (Sigma) in 200 μl of 1% ammonium bicarbonate pH 8.0 in the presence of

0.2 M sodium phosphate. Trypsin was added at an enzyme-to- substrate ratio of 1:20 (w/w) and incubation was carried out for 4 hours at 37°C.°C. Digestion was stopped by freeze- drying in Savant.

Peptides obtained by tryptic digestion were separated on a μBondapak C18 column (3.9x300 mm, lOμ Waters) or on a C4-Vydac (4.6x250 mm, 5μm) column eluted using a 60 min. linear gradient from 5-65% acetonitrile in 0.1% TFA, at a flow rate of 1.0 ml/min. Eluted peaks were manually collected, concentrated in Savant and then subjected either to amino acid analysis and to N-terminal sequence analysis on a pulsed liquid-phase mod.477A Sequencer (Applied Biosystems) . The results of C4-HPLC peptide mapping of trypsin- digested PE-P1 (A) and PE-P2 (B) are shown below.

Fragment Amino acid sequence

A 1-13 VSYTDCTESGQNY (SEQ ID NO . 6)

14-26 CLCVGGNLCGGGK (SEQ ID NO 7)

27-36 HCEMDGSGNK (SEQ ID NO 8)

37-47 CVDGEGTPKPK (SEQ ID NO 9)

37-47(*) CVDGEGX*PKPK (SEQ ID NO 10)

48-64 SQTEGDFEEIPDEDILN (SEQ ID NO 11)

B 1-13 VSYTDCTESGQNY (SEQ ID NO: 12) 14-26 CLCVGSNVCGEGK (SEQ ID NO: 13) 27-47 NCQLSSSGNQCVHGEGX*PKPK (SEQ ID NO: 14) 48-64 SQTEGDFEEIPDEDILN (SEQ ID NO: 15)

(*) X = residue not detected by amino acid sequencing; X = T by amino acid analysis.

The complete amino acid sequences of PI and P2 are therefore: PI (SEQ ID NO: 1)

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu Cys Val Gly Gly Asn Leu Cys Gly Gly Gly Lys His Cys Glu Met Asp Gly Ser Gly Asn Lys Cys Val Asp Gly Glu Gly Thr Pro Lys Pro Lys Ser Gin Thr Glu Gly Asp Phe Glu Glu lie Pro Asp Glu Asp lie Leu Asn P2 (SEQ ID NO: 2)

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu Cys Val Gly Ser Asn Val Cys Gly Glu Gly Lys Asn Cys Gin Leu- Ser Ser Ser Gly Asn Gin Cys Val His Gly Glu Gly Thr Pro Lys Pro Lys Ser Gin Thr Glu Gly Asp Phe Glu Glu lie Pro Asp Glu Asp lie Leu Asn

Example 3: Chemical synthesis of the gene coding for the analogue P2,^-Val-Glv-OH As already said, the analogue P2,. ₆₃ -Val-Gly-OH is one of formula (I) wherein P is the antithrombin polypeptide P2, Xaa is Val and Z is Gly-OH.

The nucleotide sequence coding for the analogue P2,. ₆₃ - Val-Gly-OH was designed on the basis of the Escherichia coli preferred codons". Moreover, a Ball restriction site was engineered very close to the 5' end of the synthetic P2,^ ₃ - Val-Gly-OH coding sequence to allow insertion of such sequence in different expression vectors. Indeed, the same synthetic gene was used for expression of recombinant P2,. ₆₃ - Val-Gly-OH protein in bacterial and insect cells. All plasmid DNA manipulations were carried out as described by Maniatis et al ¹² . In the case of insect cells methods were developed which yielded protein P2,. ₆₃ -Val-Gly-OH as a secreted or cytoplasmic product. In the case of bacterial cells, methods were developed to obtain secretion to the periplasm of the recombinant product. In both cases, when a secreted product is to be obtained, it is necessary to synthesize the P2,. ₆₃ Val- Gly-OH molecule 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 NH ₂ end of P2, _.ω -Val-Gly-0H ^{I I4} . This extra sequence is then cleaved off, in vivo, during secretion, by a specific leader peptidase, yielding the correct mature sequence ¹⁵ . In the insect cells expression system, we used the leader peptide of the Vescicular Stomatitis Virus (VSV) G protein, as will be described in detail in Example 5. In the case of E. coli many examples of secretion systems have been described in the literature ¹⁶¹⁷ . Among them, we have selected the system based on the secretion signal of the Outer Membrane Protein of E. coli (Omp A) previously

published ¹⁸

In order to prepare a suitable source of the P2,. ₆₃ -Val-

Gly-OH gene for the expression vectors used in the Examples, six synthetic complementary oligonucleotides were synthetized using an automated DNA synthetizer (Applied Biosystems) and their sequence is shown in Figure 3.

We designed the first two 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 RNA ¹⁹

Their sequence, shown in Fig.. 3 as oligos 1 and 2, includes also the beginning of the P2,. ₆₃ -Val-Gly-OH gene coding for the first 10 amino acids. Following enzymatic phosphorylation the six oligos were assembled using DNA ligase and the resulting double-strand sequence was inserted in the M13 phage vector mpl8, (Yanisch-Perron et al.. Gene

33. 103-119, 1985) obtaining the recombinant plasmid OMP-P2VG which is shown in Figure 4. In order to enable insertion of the P2,. ₆₃ -Val-Gly-OH gene in the M13 vector, Hindlll and BamHI 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 DNA ²⁰ .

Example 4: Expression and secretion of P2, ₆₃ -Val-Glv-OH from

E. Coli cells

From OMP-P2VG the P2, _.63 -Val-Gly-OH gene can be excised as a Hindlll-BamHI fragment which codes for the OmpA Shine- Dalgarno and leader peptide followed by the P2,. ₆₃ -Val-Gly-OH coding sequence. This restriction fragment is now ready to be inserted in an appropriate expression vector. Several expression systems could, theoretically, be err.ployed to obtain high level production of heterologous proteins in bacteria. The system based on the promoter P, has been used with success in our

laboratory in the past ¹⁹ . Again, even in the case of the selected promoter, the levels of expression of a given polypeptide cannot be predicted. Plasmid pFC33, shown in Figure 5, has already been described in the literature ¹⁹ . It carries the resistance to the antibiotic ampicillin and the bacterial promoter P^ 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-P2VG coding for the P2,. ₆₃ -Val-Gly-OH gene. The details of this construction are shown in Figure 5. We isolated a new plasmid, named pFC-P2VG, which is the final plasmid for the production of P2,. ₆₁ -Val-Gly-OH in E. coli.

An object of the present invention is the use of Ji. coli strains of the type B for the expression and secretion to the periplasm of P2,. ₆₃ -Val-Gly-OH and the other anti-thrombin analogues of the invention. Indeed, we have found that insertion of plasmid pFC-P2VG in type B strains of the bacterium E. coli brings high level production of P2 _l . ₆₃ -Val- Gly-OH. 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 P2,. ₆₃ -Val-Gly-OH.

Several type B strains of E. coli are available and can be used for the production of P2,. ₆₃ -Val-Gly-OH. Preferred strains are ATCC 12407, ATCC 11303, NCTC 10537. Below is an example of transformation of strain NCTC 10537 with plasmid pFC-P2VG and subsequent cultivation of the transformant. Competent cells of strain NCTC 10537 were prepared using the calcium chloride procedure of Mandel and Higa ²¹ . Approximately 200μl of a preparation of these cells at 1 x 10 ⁹ 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 P2,. ₆₃ -Val-Gly-OH protein 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 at37°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 HC1 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-2238 ²² , we have measured the presence of anti- thrombin activity in the periplasmic fraction of P2,. ₆₃ -Val- Gly-OH producing cells but not in control periplasmic fractions.

With the similar approach we have also constructed a new expression/secretion plasmid for P2,. ₆₃ -Val-Gly-OH where the promoter P _|pplaι . ¹⁷ is present instead of the promoter P^. This different plasmid, named pOMPA-P2VG, is shown in Figure 6. Following insertion of this plasmid in E. coli strains of the type B, high levels of active P2,. _ή3 -val-Gly-OH were also obtained. As starting plasmid for the construction of pOMPA- P2VG we used the plasmid pIN-III-ompA3 described by Ghrayb et al ²³ . Conditions for cultivation and induction of expression

with isopropyl-β-D-thiogalactopyranoside (IPTG) were as previously described ²³ .

Example 5: Expression and secretion of protein P2 _] ^-Val-Gly- OH from insect cells

To obtain secretion of protein P2 _! . ₆₃ -val-Gly-OH from recombinant insect cells we had to join the P2,. ₆₃ -Val-Gly-OH 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 protein ²⁴ . Similarly to what is described above, a synthetic DNA sequence coding for the VSVG protein leader peptide followed by the beginning of the P2,. ₆₃ -Val-Gly-OH gene has been prepared and the nucleotide sequence is given in Figure 7. Also in this case we provided convenient restriction sites (Hindlll, BamHI and Ball) to allow joining to the rest of the P2,^ ₃ -Val-Gly-OH gene and to the expression vector.

The synthetic Hindlll-Ball fragment was joined to apurified Ball-BamHI fragment from OMP-P2VG carrying the

P2,^ ₃ -Val-Gly-OH gene and inserted in M13mpl8 previously cut with Hindlll and BamHI. This construction which yielded a new plasmid named VSV-P2VG is schematically shown in Figure 8. From VSV-P2VG we have excised a BamHI-BamHI DNA fragment carrying the P2,. ₆₃ -Val-Gly-OH gene fused to tne VSV leader peptide which was then inserted into the vector pAcYMl ²⁵ , as shown in Figure 9. The resulting plasmid was named pAc-P2VG.

To obtain expression in insect cells, the VSV-P2VG 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-P2VG. 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 al ²⁶ . 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 10 ⁶ S. frugiperda 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. frugiperda 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 P2,. ₆₃ -Val-Gly-OH 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 procedures ²⁶ , 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 methods ²⁶ . In both conditions, the S-2238 chromogenic assay showed the presence of an anti-thrombin activity in the culture supernatants of the infected cells.

Example 6: Expression of protein P2, ₆₃ -Val-Glv-OH in the cytoplasm of insect cells

Protein P2, _.63 -Val-Gly-OH could also be produced and accumulated in the cytoplasm of S. frugiperda 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 protein P2^-Val-Gly-OH 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 P2,_ ₆₃ -Val-Gly-OH. The presence of the NH ₂ end sequence of polyhedrin allows high level expression ²⁷ . In addition, between the polyhedrin portion and the P2,. ₆₃ -Val-Gly-OH sequence we put a methionine residue which allows the release of the P2,. ₆₃ -Val-Gly-OH 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 OMP-P2VG to an appropriate transfer vector. The new synthetic piece, shown in Figure 10, 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 11) . Briefly, the EcoRV-BamHI fragment of pAcYMl ²⁵ 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 P2,_ ₆₃ -Val-Gly-OH coding sequence according to a scheme illustrated in Figure 12. Through this construction, we obtained a new plasmid, named pAcFTl-P2VG, which has been used to transfer the hybrid gene to the baculovirus genome.

The recombinant baculovirus was obtained as described in Example 5. Infection of S. frugiperda cells was carried out according to standard procedures ²⁶ . Cultivation of infected insect cells leads to the cytoplas ic accumulation of the fusion protein. This hybrid protein was the source of

recombinant protein P2^-Val-Gly-OH. Several methods are available from the literature which can be used to cleave the hybrid with CNBr ^28"29 . The application of the method of Olson et a_l has allowed us to obtain the correct polypeptidic sequence of P2 _! . ₆₃ -Val-Gly-OH. This molecule displayed anti- thrombin activity. Example 7

Expression and secretion in E. coli and insect cells of the analogue Pl, _.63 -Val-Gly-OH can be achieved following procedures analogous to those described above under examples 3 to 6. For this purpose, six synthetic complementary oligonucleotides were prepared and their sequence is shown in Figure 13. Oligos 1,2,5 and 6 are identical to those used for the genetic construction of Figure 3, since the differences in the sequence of Pl,. ₆₃ -Val-Gly-OH and P2,. ₆₃ -Val-Gly-OH are in the central part of the molecule and the corresponding codons are located in oligos 3 and 4.

Example 8

The analogue P, _.63 -Val-Gly-OH is converted to the corresponding amidated product P,. ₆₃ -Val-NH ₂ by incubation with a partially purified protease-free PAM enzyme preparation, as, for example, obtained from rat thyroid medullary carcinoma ³⁰ or from the conditioned medium of cells derived from the same tumour ³¹ .

Alternatively, recombinant DNA techniques can also be applied to prepare an amidating enzyme.

The enzymatic amidating reaction has been carried out in an aqueous buffer, for example TSE buffer, supplemented with copper ions, ascorbate, catalase, potassium iodide, SDS and

Tween 20.

The progress of the amidation reaction can be monitored by the method of Corbett and Corbett ³² which detects the formation of glyoxylic acid after derivatization with nitrosobenzene.

The amidation product P,. ₆₃ -Val-NH ₂ can be purified according to standard techniques well known to the skilled in the art. Example 9 Biological activity of P2, ₆₃ -Val-Gly-OH Antithrombin activity

The antithrombin activity of the analogues P2 ₁ . ₆₃ -Val-Gly-OH of the present invention was determined on the basis of the rapid and stoechiometric reaction of the analogue peptides with thrombin.

This activity was measured quantitatively by means of titration of a standard solution of thrombin ³³ . Thrombin activity was calibrated with the International Standard Preparation of Thrombin (c 70/157) obtained from the National Institute for Biological Standards and Control (London, U.K.) and expressed in National Institute of Health Units (N.I.H. units) .

The thrombin neutralizing activity of the samples was expressed as antithrombin units (ATU) ; one ATU is the amount of test compound which neutralizes an NIH unit of thrombin. Recombinant hirudin (HVl variant; rHVl) and recombinant P2 protein (rP2) were considered as reference compounds. The test was performed as follows: 0.2 ml of a standard solution of 0.05% human fibrinogen (Kabi Vitrum, Sweden) in Tris HCl buffer pH 7.4 were incubated at 37°C in the presence of 0.01 to 0.1 ml of the solutions of P2^-Val-Gly-OH.

Thereafter, aliquots of 0.005 ml (0.5 NIH units) of a 100 NIH/ml thrombin standard solution were added progressively each minute and mixed gently.

The end point of the titration was considered to be reached when a fibrin clot was formed within one minute. Indeed, clot formation only occurred when a sufficient amount of thrombin was added to the reaction mixture, the added thrombin being able to neutralize the total amount of the analogue present in the mixture.

The antithrombin activity of the analogue P2,. ₆₃ -Val-Gly-OH has been expressed in ATU/mg protein (see table 1) .

Protein content was determined by aminoacid analysis. Chromogenic substrate assay

The principle of the method is based on the inhibitory activity of P2, _.63 -Val-Gly-OH on the reaction between thrombin and its specific chromogenic substrate S-2238 ³⁴ .

Recombinant hirudin HV1 and recombinant P2 protein were used as references.

The test was performed as follows:

630 μl of a 0.5 mM solution of S-2238 in 0.05 M Tris buffer pH 8.3 containing 0.IN NaCl and 100 KlU/ml aprotinin were pre-incubated for 10 min. at 25°C. After this pre-incubation period, 70 μl of a 2 IU/ml thrombin solution in 0.025 M sodium phosphate buffer pH 6.6 containing 0.3 M NaCl and 0.5% bovine albumin were added.

For the controls, 70 μl of buffer were used.

Immediately after the addition of thrombin, the absorbance variations were recorded spectrophotometrically at a wave length of 405 nm.

Recordings were made kinetically each minute over a total period of 6 minutes.

Thereafter, 20 μl of the solutions of the tested analogue were added and absorbance variations were recorded again as described before.

Final concentrations of the test compounds were 1.25, 2.5,

7.5, 10 and 15 ng/ml in 0.025 M sodium phosphate buffer pH

6.6 containing 0.3 M NaCl. The absorbance variations per minute (Δ Abs/min) were determined respectively before and after the addition of the sample and the mean Δ Abs/min. were calculated.

The inhibition of the reaction between thrombin and the chromogenic substrate S-22?^ was calculated as the ratio (expressed as percentage) between the mean Δ Abs/min. before and after the addition of the analogue.

A standard curve has been established on the basis of the percent inhibition versus the concentration of the sample.

Final results were expressed as final concentrations of the test compounds inducing 50% inhibition of the reaction between thrombin and its chromogenic substrate.

The thrombin time

The antithrombin activity of the analogue P2,. ₆₃ -Val-Gly-OH has been determined by measuring the thrombin time in human plasma.

Recombinant hirudin HV1 (rHVl) and recombinant P2 protein

(rP2) wre used as references.

The test was performed as follows:

Mixtures of normal human citrated plasma containing increasing final concentrations of the tested analogue or the reference compounds were placed in an automatic coagulometer

(ACL 300 research, Instrumentation Laboratory, Italy) , and human thrombin (Fibrindex, Ortho Diagnostics, Italy: 5 IU/ml final concentration) was added. The clotting times were recorded and plotted against the final concentration of each sample.

Final results were expressed as the final concentration of each compound required to double the normal value of the thrombin time (ratio=2) . In all the test performed, the antithrombin activity of P2,. ₆₃ -

Val-Gly-OH resulted considerably superior to that of the reference compounds.

Table 1 reports the data obtained with the analogue P2,. ₆₃ -Val-

Gly-OH in the biological assays discussed above.

TABLE 1

Compound ATU/mg S-2238 Thrombin

IC50 Time in ng/ml in μg/ml

rHVl 11.000 5.47 ratio=2

at 0.132 μg/ml rP2 11.900 5.43 ratio=2 at 0.131 μg/ml

P2 _{1 B} -Val- 20.000 0.6 ratio=2 Gly-OH at 0.047 μg/ml

References

1) Markwardt, F. 1970, Methods in Enzy ology, JL9_, P- 924

2) Markwardt, F. 1985, Biomed. Biochim. Acta. 44./ P» 1007 3) Markwardt, F. Hauptmann, J. , Nowak, G. , Klessen, C. , and Walsmann, P. 1982. Thromb. Haemostasis 4_7, p. 226

4) A.F. Bradbury et al., 1982, Nature, 289: 686-688

5) G.S. Wand et al., 1985, Neuroendocrinol, 4JL, 482-489

6) B. EIPPER et al. 1983, Proc.Natl.Acad.Sci: USA 80, 5144- 5148

7) Breddam, K. et al. Int. J. Pept. Prot. Res.: 21_, 153- 160, 1991;

8) Henriksen, D.B. et al. J. Am. Chem. Soc. : 114 , 1876- 1877, 1992 9) Lundblad, R.L., 1971, Biochemistry, 10: 2501-2506

10) Dupont D., Keim P., Chui A., Bello R. and Wilson K. , Derivatizer-Analyser User Bulletin No. 1, Applied Biosystems Inc., 1987

11) Grosjeans H. and Fiers W. 1982. Gene, 18 _. , p. 199 12) Maniatis T. , Fritsch E.F. and Sambrook J. 1982. Cold Spring Harbor, NY

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

14) Pages J.M. 1983, Biochimie, 6_5, p. 531 15) Wolfe P.B. 1983. J. Biol. Chem. 258. p. 12073

16) Talmadge K. , Stahl S. and Gilbert W. 1980. Proc. Natl. Acad. Sci. USA, 7_7, p. 3369

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19) Isacchi A., Sar ientos P., Lorenzetti R. and Soria M. 1989, Gene 8 _. 1, p. 129

20) Sanger, F., Nicklen, S., and Coulson, A.R. 1977, Proc.

Natl. Acad. Sci. USA 7_4, p. 5463.

21) Mandel M. and Higa A.J. 1970. J. Mol. Biology, 5_3, p.154

22) Krstenansky, J.K., and Mao, S.J.T. 1987. FEBS Lett. 211. p. 10

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24) Bailey, M.J. , McLeod, D.A. , Kang, C. , and Bishop, D.H.L. 1989. Virology 169. p. 323 25) Matsuura, Y., Possee, R.D., Overton, H.A. and Bishop. D.H.L. 1987. J. Gen. Virol. 68, P- 1233

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34) Wiman et al., BBA, 579. 142-154, 1979

SEQUENCE LISTING

(1) INFORMATION FOR SEQ ID NO: l :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 64 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu Cys 1 5 10 15

Val Gly Gly Asn Leu Cys Gly Gly Gly Lys His Cys Glu Met Asp Gly 20 25 30

Ser Gly Asn Lys Cys Val Asp Gly Glu Gly Thr Pro Lys Pro Lys Ser 35 40 45

Gin Thr Glu Gly Asp Phe Glu Glu l ie Pro Asp Glu Asp lie Leu Asn 50 55 60

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 64 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu Cys 1 5 10 15

Val Gly Ser Asn Val Cys Gly Glu Gly Lys Asn Cys Gin Leu Ser Ser 20 25 30

Ser Gly Asn Gin Cys Val His Gly Glu Gly Thr Pro Lys Pro Lys Ser 35 40 45

Gin Thr Glu Gly Asp Phe Glu Glu lie Pro Asp Glu Asp lie Leu Asn 50 55 60

(3) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr Cys Leu Cys 1 5 10 15

Val Gly Ser Asn Val Cys Gly Glu Gly Lys Asn Cys Gin Leu Ser Ser 20 25 30

Ser Gly Asn Gin Cys Val His Gly Glu Gly 35 40

(4) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE: (A) ORGANISM: E. coli

(ix) FEATURE:

(A) NAME/KEY: misc eature

(B) LOCATION: 1..63

(D) OTHER INFORMATION: /function = "leader peptide" /standard_name= "OmpA leader"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

ATGAAAAAGA CAGCTATCGC GATTGCAGTG GCACTGGCTG GTTTCGCTAC

CGTAGCGCAG GCC 63

(5) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: vesicular stomatitis virus

(ix) FEATURE:

(A) NAME/KEY: misc eature

(B) LOCATION: 1..48

(D) OTHER INFORMATION: /function = "leader peptide" /standard_name= "VSV G protein leader"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

ATGAAGTGCC TTTTGTACTT AGCCTTTTTA TTCATTGGGG TGAATTGC 48

(6) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(B) LOCATION: 1..13

(D) OTHER INFORMATION: /note= "This sequence is amino acids 1..13 of seq id no: l "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr 1 5 ^" 10

(7) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(B) LOCATION: 1..13

(D) OTHER INFORMATION: /note= "This sequence is amino acids 14..26 of seq id no: l "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Cys Leu Cys Val Gly Gly Asn Leu Cys Gly Gly Gly Lys 1 5 10

(8) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(B) LOCATION: 1..10

(D) OTHER INFORMATION: /note= "This sequence is amino acids 27..36 of seq id no: l "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

His Cys Glu Met Asp Gly Ser Gly Asn Lys 1 5 10

(9) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(B) LOCATION: 1..1 1

(D) OTHER INFORMATION: /note= "This sequence is amino acids 37..47 of seq id no: l "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Cys Val Asp Gly Glu Gly Thr Pro Lys Pro Lys 1 5 10

(10) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1 1 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(B) LOCATION: 1..11

(D) OTHER INFORMATION: /note= "This sequence is amino acids 37..47 of seq id no: l "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Cys Val Asp Gly Glu Gly Xaa Pro Lys Pro Lys 1 5 10

(11) INFORMATION FOR SEQ ID NO: 1 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1.. 16

(D) OTHER INFORMATION: /note= "amino acids 48..64 of seq id no: l "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 1 :

Ser Gin Thr Glu Gly Asp Phe Glu Glu l ie Pro Asp Glu Asp lie Leu 1 5 10 15

Asn

(12) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(B) LOCATION: 1..13

(D) OTHER INFORMATION: /note= "This sequence is amino acids 1..13 of seq id no:2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Val Ser Tyr Thr Asp Cys Thr Glu Ser Gly Gin Asn Tyr 1 5 10

(13) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hiudinaria manillensis

(ix) FEATURE:

(B) LOCATION: 1..13

(D) OTHER INFORMATION: /note= "This sequence is amino acids 14..26 of seq id no:2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Cys Leu Cys Val Gly Ser Asn Val Cys Gly Glu Gly Lys 1 5 10

(14) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1..21

(D) OTHER INFORMATION: /note= "This sequence is amino acids 27..47 of seq id no:2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Asn Cys Gin Leu Ser Ser Ser Gly Asn Gin Cys Val His Gly Glu Gly 1 5 10 15

Xaa Pro Lys Pro Lys 20

(15) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(ix) FEATURE:

(A) NAME/KEY: Region

(B) LOCATION: 1..21

(D) OTHER INFORMATION: /note= "This sequence is amino acids 48..64 of seq id no:2"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

Ser Gin Thr Glu Gly Asp Phe Glu Glu l ie Pro Asp Glu Asp lie Leu 1 5 10 15

Asn

(16) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 198 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GTTTCTTACA CCGACTGCAC CGAATCTGGC CAGAACTACT GCCTGTGCGT 50

TGGTTCTAAC GTTTGCGGTG AAGGTAAAAA CTGCCAGCTG TCTTCTTCTG 100 GTAACCAGTG CGTTCACGGT GAAGGTACCC CGAAACCGAA ATCTCAGACT 150

GAAGGTGACT TCGAAGAAAT TCCGGACGAA GACATCCTGG TTGGTTAG 198

(17) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 198 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GTTTCTTACA CCGACTGCAC CGAATCTGGC CAGAACTACT GCCTGTGCGT 50

TGGTGGTAAC CTGTGCGGTG GTGGTAAACA CTGCGAAATG GATGGTTCTG 100

GTAACAAATG CGTTGATGGT GAAGGTACCC CGAAACCGAA ATCTGAGACT 150

GAAGGTGACT TCGAAGAAAT TCCGGACGAA GACATCCTGG TTGGTTAG 198

(18) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vii) IMMEDIATE SOURCE: (B) CLONE: oligo 1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

AGCTTTGATA ACGAGGCGCA AAAAATGAAA AAGACAGCTA TCGCGATTGC 50

AGTGGCACTG GCTGGTTTCG CTACCGTAGC GCAGGCCGTT TCTTACACCG 100

ACTGCACCGA ATCTGG 116

(19) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 2

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AACTATTGCT CCGCGTTTTT TACTTTTTCT GTCGATAGCG CTAACGTCAC 50 CGTGACCGAC CAAAGCGGTG GCATCGCGTC CGGCAAAGAA TGTGGCTGAC 100 GTGGCTTAGA CCGGTCT 1 17

(20) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 88 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CCAGAACTAC TGCCTGTGCG TTGGTTCTAA CGTTTGCGGT GAAGGTAAAA 50 ACTGCCAGCT GTCTTCTTCT GGTAACCAGT GCGTTCAC 88

(21) INFORMATION FOR SEQ ID NO:21 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 87 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21 : TGATGACGGA CACGCAACCA AGATTGCAAA CGCCACTTCC ATTTTTGACG 50 GTCGACAGAA GAAGACCATT GGTCACGCAA GTGCCAC 87

(22) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 85 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 5

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GGTGAAGGTA CCCCGAAACC GAAATCTCAG ACTGAAGGTG ACTTCGAAGA 50 AATTCCGGAC GAAGACATCC TGGTTGGTTA GTAAG 85

(23) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 85 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 6

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TTCCATGGGG CTTTGGCTTT AGAGTCTGAC TTCCACTGAA GCTTCTTTAA 50 GGCCTGCTTC TGTAGGACCA ACCAATCATT CCTAG 85

(24) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 91 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: vesicular stomatitis virus

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 1 VSV

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: AGCTTGGATC CACTATGAAG TGCCTTTTGT ACTTAGCCTT TTTATTCATT 50 GGGGTGAATT GCGTTTCTTA CACCGACTGC ACCGAATCTG G 91

(25) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 87 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: vesicular stomatitis virus

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 2 VSV

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: CCAGATTCGG TGCAGTCGGT GTAAGAAACG CAATTGACCC CAATGAATAA 50 AAAGGCTAAG TACAAAAGGC ACTTCATAGT GGATCCA 87

(26) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 1 fusion

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GATCCATGGT TTCTTACACC GACTGCACCG AATCTGG 37 (27) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE: (B) CLONE: oligo 2 fusion

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CCAGATTCGG TGCAGTCGGT GTAAGAAACC ATG 33

(28) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 150 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: baculovirus

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 1 polyhedrin

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

ATCATGGAGA TAATTAAAAT GATAACCATC TCGCAAATAA ATAAGTATTT 50

TACTGTTTTC GTAACAGTTT TGTAATAAAA AAACCTATAA ATATGCCGGA 100

TTATTCATAC CGTCCCACCA TCGGGCGT C CTACGTGTAC GACAACACCG 150

(29) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 154 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: baculovirus

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 2 polyhedrin

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GATCCGGTGT TGTCGTACAC GTAGGTACGC CCGATGGTGG GACGGTATGA 50 ATAATCCGGC ATATTTATAG GTTTTTTTAT TACAAAACTG TTACGAAAAC 100 AGTAGAATAC TTATTTATTT GCGAGATGGT TATCATTTTA ATTATCTCCA 150 TGAT 154

(30) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 88 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

CCAGAACTAC TGCCTGTGCG TTGGTGGTAA CCTGTGCGGT GGTGGTAAAC 50 ACTGCGAAAT GGATGGTTCT GGTAACAAAT GCGTTGAT 88

(31) INFORMATION FOR SEQ ID NO:31 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 87 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(vii) IMMEDIATE SOURCE:

(B) CLONE: oligo 4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31 : TGATGACGGA CACGCAACCA CCATTGGACA CGCCACCACC ATTTGTGACG 50 CTTTACCTAC CAAGACCATT GTTTACGCAA CTACCAC 87

32) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Met Phe Ser Leu Lys Leu Phe Val Val Phe Leu Ala Val Cys lie Cys 1 5 10 15

Val Ser Gin Ala 20

(33) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 204 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Hirudinaria manillensis

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

GTTTCTTACA CCGACTGCAC CGAATCTGGC CAGAACTACT GCCTGTGCGT 50

TGGTTCTAAC GTTTGCGGTG AAGGTAAAAA CTGCCAGCTG TCTTCTTCTG 100

GTAACCAGTG CGTTCACGGT GAAGGTACCC CGAAACCGAA ATCTCAGACT 150

GAAGGTGACT TCGAAGAAAT TCCGGACGAA GACATCCTGA ACGGTGCTTA 200 GTAA 204