CA2318358A1 - Polypeptides, dna coding therefor, formulations thereof, and their use in inhibiting factor xii activation - Google Patents

Abstract

A polypeptide, derivable from a Haementeria leech, which polypeptide is suitable for inhibiting C1 esterase and/or factor XII activation is disclosed, together with a nucleic acid sequence encoding the polypeptide. The polypeptide may be used as a pharmaceutical formulation or in gene therapy to treat or prevent cardiovascular, inflammatory or auto-immune disorders.

Description

Polypeptides, DNA Coding therefor, Formulations thereof, and their Use in Inhibiting Factor XII Activation The present invention relates to a novel class of inhibitors of the initiation steps of blood coagulation and complement that can be derived, for example, from leech tissues or secretions. In particular, the present invention relates to polypeptides, cDNA
encoding therefor, and the use of the polypeptides and formulations thereof in cardiovascular, auto-immune and inflammatory disease.
The blood coagulation system is a cascade of enzymes that results in the formation of fibrin, which is an insoluble, fibrous polymeric protein that plugs leaks in the vasculature and prevents blood loss. The individual enzymes of the coagulation system circulate in the normal state as pro-enzymes (so-called 'zymogens'), v~r~ch are normally inactive.
The system can become activated by either:
(a) contact with negatively-charged foreign surfaces, such as glass or some materials fmm which prosthetic device w .. ~~~~d. This is the so-called 'intrinsic syste~,;;'; or (b) binding of factor VII to tissue factor, which is a membrane protein that is expressed on the outer surface of many cell types, particularly those found in blood vessel walls and that come into contact with blood at sites of leakage.
This is called the 'extrinsic system'.
The first phase of the intrinsic pathway involves the binding of factors XII, XI, prekallilaein and high molecular weight kininogcn in a complex on the negatively-charged surface. Through interacting events within the complex, factor XII
becomes activated to factor aJCIa which, in turn, activates factor XI to XIa. The whole process is accelerated by the activation of prekallilQCin to kallil~ein. Factors a.3~a, ~i~ia, XIa ~d kallikrein are all serine proteases of the initiation complex.
Like all physiological mechanisms, the coagulation cascade can become activated inappropriately and result in the formation of haemostatic plugs inside the blood vessels.

WO 99/3b439 PCT/GB99/00147 z Thereby, vessels can become blockerd and the blood supply to distal organs limited. This process is known as thrombosis and is associated with high mortality. In addition, the use of prosthetic devices that ~ are in contact with blood is severely limited because of activation of the coagulation cascade and coating of the prosthetic surface, often compromising its function. Examples of such prosthetic devices are haemodialysers, cardiopulmonary by-pass circuits, vascular stents and in-dwelling catheters.
In cases where such devices are used, anticoagulants, such as heparin, are used to prevent fibrin from depositing on the surface. However, some patients are intolerant of heparin, which can cause thrombocytopaenia and the resultant risk of serious bleeding. There is therefore a need for new types of anticoagulant that do not cause such problems and that can be used in affected patients.
The complement system in mammalian blood is involved in the defence of the body against infection by foreign organisms. Like the coagulation system, it is also a complex enzyme cascade. When activated, it leads to the clearance of invading organisms, either indirectly, by coating them with proteins that are recognised by phagocytic cells (so-called 'immune adherence) or directly, by lysis. It also causes a localised inflammatory response leading to leukocyte activation and migration; increased vascular permeability;
contraction of smooth muscle; and release of biogenic amines.
Activation of complement can occur through one or both of two pathways: (a) the classical pathway; and (b) the alternative pathway:
(a) The first part of the classical complement pathway is analogous to the coagulation cascade. Activation can take place on an antibody-coated surface or other negatively-charged surface and gives rise to a serine protease, C 1 esterase.
This, in the presence of an activated cofactor (C4b), specifically cleaves the next protease zymogen (C2) in the cascade and leads eventually to an end-product that, in this case, is the "membrane attack complex", which lyses the targeted cell.
The pathway also leads to the production of various by-products, which are themselves biologically active and act to potentiate the inflammatory response.
(b) For the alternative pathway, the stimulus can be bacterial lipopolysaccharides;
various polyanions, the FAb portions of immunoglobulins; and negatively charged phospholipids. It lead's to direct activation of C3 without involving C1, C2 or C4.
C1 esterase is therefore the activated initiation complex of the classical complement pathway. The first component of the classical pathway, C1, is a heteropolymer comprising one molecule of C 1 q, and two each of C lr and C 1 s. C 1 q binds either to the Fc portion of either IgG or IgM or to negatively-charged substances, such as DNA, carrageenan, heparin, dextran sulphate, chondroitin sulphate, certain bacterial lipopolysaccharides and viral envelopes. After binding, the Clq induces the autocatalytic activation of C lr to a serine protease, which in turn activates C 1 s to another serine protease. The activated C 1 s in the complex can cleave both C4 and C2, which is essential in the activation of the whole classical pathway.
As with the coagulation cascade, the complement cascade can also be activated inappropriately, and in such circumstances causes marked inflammation. It is thereby involved in the pathology of a large number of inflammatory and auto-immune diseases.
It is therefore desirable to provide new inhibitors of the complement pathway for the treatment of such diseases; to reduce tissue rejection of implanted organs;
and to inhibit complement activation on foreign surfaces, such as haemodialysers and cardiopulmonary by-pass circuits.
It is therefore an object of this invention to provide inhibitors of the initiation complexes of the intrinsic coagulation cascade and/or of the classical complement cascade, and thereby to reduce the incidence of inappropriate clotting on negatively-charged surfaces and/or the production of pharmacologically-active complement components. Such inhibitors are expected to have an advantage over inhibitors that act on enzymes lower down the cascades, because the initiation complexes occur in much lower concentrations.
This enables them to be inhibited by lower concentrations of their inhibitors, which could then be administered in lower doses, avoiding possible toxicity and reducing the cost of treatment, relative to inhibitors of enzymes lower down the cascades.
Known inhibitors of satins proteases in the initiation complexes of coagulation and complement fall into three categories: low molecular weight amidines or guanidines;
small polypepddes that can be isolated from parasites; and the naturally-occurring blood serpin (satins protease inhibitor), C 1 inhibitor.
All three exhibit disadvantages in terms of their potential for use in medical treatments:
The low molecular weight compounds described to date lack specificity and inhibit factor XIIa and C 1 esterase much less potently than other proteases. Therefore, they are not suitable for treating human complement-mediated disease.
Although some small polypeptides inhibit some satins pmteases, none so far has been demonstrated to inhibit the initiation complexes of either the classical pathway of complement activation or the intrinsic pathway of blood coagulation.
However, the plasma serpin, C1 inhibitor, inhibits both the intrinsic activation of coagulation and C 1 esterase, although it is rather non-specific, as it inhibits factors XIIa, XIa, kallikrein, C 1 s and C 1 r. C 1 inhibitor can be purified from human blood and a recombinant version has also been described. C 1 inhibitor from blood is used to treat the disease of its own deficiency, angioneurotic oedema. Recent evidence in pilot human clinical trials indicates that it i~ also effective ~.~. Lreating both septic shock and capillary leak syndrome. Fu.~er the administration of C1 inhibitor in a feline model of myocardial reperfus:~n injury significantly improved the recovery of cardiac contractility and reduced the infarcted area of the myocardium. C1 inhibitor is a large protein of molecular weight 104,000 Da, containing 478 amino acids; the recombinant pmtein is consequently very expensive to produce. In view of the promising effects of C1 inhibitor in the clinic, but its production difficulties, there is a need for new compounds that mimic the effects of C1 inhibitor but which are easier and less expensive to produce.
It is therefore a further object of the present invention to pmvide inhibitors of the initiation complexes of the coagulation and/or, especially, the complement cascade, which are more suitable for pharmaceutical production and/or therapeutic use. To this end, we have now isolated from ectoparasitic leeches of the genus Haementeria and characterised new polypeptides that inhibit the initiation complexes of both coagulation and/or complement. These polypeptides have an electrophoretic mobility on SDS PAGE
consistent with an apparent molecular weight of between 20 and 25 kDa and have the following ten amino acids at the N-terminus;
X-K-K-K=L-P-K-X'-Q-K- (SEQ ID No: 1]
5 wherein amino acids are.abbreviated to their single letter codes; X
represents any natural amino acid, preferably alanine or N-acyl alanine or N-alkyl alanine, especially alanine;
and X' may be cysteine [SEQ 1D No: 9] or glutamic acid [SEQ )D No: 10], especially cysteine. By 'acyl' is meant (C,_5 allcoxy)carbonyl, such as formyl or acetyl;
and by 'alkyl' is meant a straight or branched C,_s alkyl chain.
In particular, these polypeptides have the following ten amino acids at the N-terminus, ie wherein, in [SEQ m No: 1 ], X is alanine:
A-K-K-K-L-P-K-X'-Q-K [SEQ ID Nos: 11 and 12]
and X' is, as above, cysteine or glutamic acid, respectively, especially cysteine.
Preferably, the polypeptides according to the present invention comprise the sequence of 37 amino acids [SEQ ID No: 13]; more preferably, the sequence of 60 amino acids [SEQ
1D No: 2].
Especially preferred is when the polypeptide according to the present invention comprises the sequence of 122 amino acids [SEQ 1I7 No: 3], which exhibits a calculated molecular weight of about l4kDa:

AKKKLPKCQKQEDCGSWDLKCNNVTKKCECRNQVCGRGCP

KERYQRDKYGCRKCLCKGCDGFKCRLGCTYGFKTDKKGCE

AFCTCNTKETACVNIWCTDPYKCNPESGRCEDPNEEYEYDYE

It will be understood that [SEQ ID Nos: 1, 2 and 9 to 13J are fragments or partial sequences of [SEQ ID No: 3].
The present invention therefore provides a polypeptide, in particular an isolated, purified or synthesised polypeptide, derivable from a Haementeria leech, which polypeptide is suitable for inhibiting C1 esterase and/or factor XII activation and which polypeptide comprises a sequence of amino acids as hereinbefore defined.
The present invention further provides a polypeptide that is substantially homologous or analogous to any of the sequences specified herein, including derivatives (such as a chimeric derivative) or bioprecursors thereof (including wherein the polypeptide is linked to a so-called 'leader sequence'), or salts of any of these.
such Haementeria-derivable polypeptides encompassed within the scope of this invention are hereinafter collectively referred to as 'haemostasins'.
Haemostasins are potent inhibitors of the initiation complexes of blood coagulation and/or of complement. As a consequence, they prolong the activated partial thromboplastin clotting time of human plasma (as shown hereinafter in Example 8) and/or inhibit the haemolysis of antibody-coated sheep erythrocytes in a classical CHS°
assay (as shown hereinafter in Example 10) at concentrations in the range of from 0.01 to 5 pg/ml.
In the definition of the haemostasins of the present invention, by 'homologue' is meant a polypeptide in which no more than (5) 20% of the amino acids in the polypeptide chain differ from those listed. The figure of 20% is based on the fact that many homologues of another leech protein, hirudin, occur naturally in Hirudo medicinalir and are described in the literature; the most diverse of these differ in 1 S of the 65 amino acids in the polypeptide chain. Like hirudin, the haemostasins are polymorphic and sequences where amino acid number 15 may be threonine (T) instead of serine (S) [SEQ ID No: 7]
and where number 60 may be asparagine (1~ instead of aspartic acid (D) [SEQ ID No:
8] are encompassed.
By 'analogue' is meant that one or more additional amino acids may be interposed in the WO 99/36439 PCTlGB99/00147 polypeptide chain, provided that they do not interfere with the pharmacological activity of the haemostasin.
The haemostasins according to this invention therefore also encompass amino acid sequences including post-translational modifications, such as sulphation of the aromatic ring of tyrosines 119 and/or 121, as has been observed in the hirudins.
Furthermore, since the motif N V T occurs at positions 23 - 25 of (SEQ ID No: 2], which is a well-known site for potential glycosylation, the haemostasins further encompass polypeptides of the [SEQ
ID No: 2] where aspargine 23 is modified by an N-linked complex carbohydrate containing not more than 10 (5 10) sugars or sugar derivatives in a single or branched chain.
Thus haemostasins also encompass truncated and therefore lower molecular weight forms of the polypeptides defined above at the N-terminus, for example forms where one or two of the N-terminal amino acids, such as given in (SEQ ID No: 1 ], are deleted;
and forms where the N-terminus is extended by the addition of one or two amino acids to the N-terminal amir.~ group. Likewise, haemostasins also encompass truncated or extended (lower or higher molecular weight) forms of the polypeptide of [SEQ m No: 3, 7 or 8] at the C-terminus. In particular, haemostasins also encompass the case where the sequence is cleaved, especially after amino acid 37 (R) of [SEQ ID No: 2, 3, 7 or 8], so that it exists as two or more polypeptide chains normally cross-linked by the usual disulphide bonds.
By 'bioprecursor' is meant a polypeptide that converts to a haemostasin according to the present invention in vivo or otherwise under conditions of use. In particular, haemostasins encompass the case where a so-called 'leader sequence' is present when the polypeptide is expressed in vivo, especially the case where the leader sequence comprises [MSFKIVLLLFLVVCVVASLA].
Haemostasins can advantageously form salts, preferably pharmaceutically acceptable salts, with any suitable non-toxic metal ion, organic or inorganic acid, or base. Examples of such inorganic acids include hydrochloric, hydrobromic, sulphuric, phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrosulphate. Examples of organic acids include mono-, di- and tri-carboxylic acids, s such as acetic, gkycolic, lactic, pyruvic and sulphonic acids, or the like.
Examples of bases include ammonia, primary, secondary or tertiary amines, or quaternary ammonium ion. Other suitable salts areknown to those skilled in the art.
Haemostasins may be extracted from Haementeria leech tissue or secretions by, for example, homogenisation of substantially the whole leech, or its salivary glands, its proboscis or the like; in a suitable buffer. Neither inhibitors of factor XII
activation nor complement inhibitors have been previously identified in, or extracted from, Haementeria leeches. The present invention therefore further provides an inhibitor of factor XII
activation and/or an inhibitor of C1 esterase, derivable from Haementeria leech tissue or secretions. Alternatively or in addition, the present invention provides a haemostasin derivable from a Haementeria leech, which haemostasin is suitable for inhibiting factor XII activation and/or C 1 esterase activity. Preferably, the inhibitor or haemostasin inhibits both factor XII activation and C 1 esterase activity.
The term 'derivable', as used herein, encompasses both material that is directly derived, such as by isolation and/or purification, as well as material which is indirectly derived or converted to a chemically-modified derivative, or which is chemically or biologically synthesised, including genetically-engineered.
The inhibitors or haemostasins according to this invention are typically extracted or purified from Haementeria leeches using a combination of known techniques, such as ion-exchange, gel filtration and/or reverse phase chromatography.
Leeches of the same genus or even the same species often have polypeptides in their saliva that have similar biochemical effects and highly homologous amino acid sequences. Furthermore, in the same species of leech, several different isoforms may exist that differ by only a few amino acids. Because many of the components of the salivary gland or tissue secretions from leeches that have similar biochemical specificity are members of homologous families of polypeptides, the present invention also comprises such isoforms and analogues derivable from Haementeria leeches, as described hereinabove.
According to another aspect of the present invention, there is provided an inhibitor of the intrinsic pathway of blood coagulation, which inhibitor is derivable from leech tissue or leech secretions finm leeches of the order Rhynchobdellida, of the genus Haementeria, and especially from the species ghilianii or of, j~cinalis, more especially, H. ghilianii. Such inhibitors have an anticoagulant effect in plasma, since they prolong the activated partial thromboplastin time but not the prothrombin time or thrombin time. This effect is probably caused by the inhibitory eft'ect of haemostasins on the activation of plasma by surfaces, such as glass or dextran sulphate, that can be measured by a reduction in the appearance of enzyrnically active factor (3XIIa or factor XIa.
A further aspect of this invention provides an inhibitor of C1 esterase, which inhibitor is derivable from leech tissue or leech secretions from leeches of the order Rhynchobdellida, of the genus Haementeria, and especially from the species ghilianii or o~cinalis, more especially, H. ghilianii. Haemostasins have the ability to inhibit: the enzymic cleavage of 4-nitroanilide from d-Val-Ser-Arg-4-nitroanilide by purified Cls; the whole complement-mediated haemolysis of antibody-sensitised erythrocytes; and the formation of the immunologically-detected membrane attack complex in serum in response to bound antibodies.
Alternatively, haemostasins can also be prepared by providing a host, transformed with an expression vector comprising a DNA sequence encoding the haemostasin polypeptide under such conditions that said polypeptide is expressed therein and thereafter, if desired, isolating the polypeptide thus obtained. This approach is typically based on obtaining a nucleotide sequence encoding the polypeptide it is wished to express and expressing the polypeptide in recombinant organisms. The cultivation of the genetically-modified organism leads to the production of the desired product displaying full biological activity.
The present invention therefore also comprises a recombinant haemostasin, or a synthetic, or genetically- or protein-engineered, equivalent to a haemostasin polypeptide according to the invention.
Accordingly, the present invention fiuther provides a nucleic acid sequence, in particular an isolated, purified or recombinant nucleic acid sequence, comprising:
(a) a sequence encoding a polypeptide, particularly a haemostasin, encompassed by to the present invention; .
(b) a sequence substantially homologous to or that hybridises to sequence (a) under stringent conditions;
(c) a sequence substantially homologous to or that hybridises to the sequence (a) or (b) but for the degeneracy of the genetic code; and (d) an oligonucleotide specific for any of the sequences (a), (b) or (c).
In particular, the present invention provides a nucleic acid sequence as defined above, wherein the sequence is a DNA or RNA sequence, such as cDNA or mRNA. More particularly, the present invention provides a DNA sequence identified herein by [SEQ ID
No: 4], which sequence corresponds with the polypeptide identified herein as [SEQ m No: 3] including its leader sequence. Given the polymorphisms already described above with reference to [SEQ ID Nos: 7 and 8], the present invention further provides the corresponding DNA sequences identified herein as [SEQ >D Nos: 5 and 6], respectively.
Therefore, the present invention fitrther provides a method for the preparation of a polypeptide according to the present invention, which method comprises:
(a) isolation and/or purification of tissue or extracts from a Haementeria leech; or (b) expression of a nucleic acid sequence encoding the polypeptide and, optionally, isolation and/or purification of the resulting polypeptide.
The present invention further provides: a recombinant construct comprising any nucleic acid sequence according to the invention; a vector comprising such a construct; and a host transformed or transfected by such a vector.
The present invention therefore further provides a cell, plaslnid, virus, live organism or other vehicle that has been genetically or protein-engineered to produce a polypeptide or haemostasin according to the present invention, said cell, plasmid, virus, live organism or other vehicle having incorporated expressably therein a sequence as disclosed herein.
Such cells may include animal, such as mammal, eg human or humanised cells, for use in gene therapy to treat or prevent conditions such as those mentioned herein. In another aspect, the present invention therefore provides a method for the treatment or prevention of a condition or disorder mentioned herein, wherein the polypeptide is administered by tt means of being expressed in the cells of the patient, which cells have incorporated expressably therein a nucleic acid sequence. Alternative to gene therapy, the haemostasins of the invention may be adiriinistered as a pharmaceutical formulation.
Accordingly, the present invention provides the use of a polypeptide, particularly a haemostasin, described herein or a nucleic acid sequence coding for the polypeptide in medicine, including ~ gene therapy; and also the use of such a polypeptide in the manufacture of a medicament.
Therefore, according to a fitrther aspect of the present invention, there is provided a pharmaceutical formulation comprising a haemostasin according to the invention (as described above) and a pharmaceutically acceptable carrier therefor. The term "phatinaceuticaliy acceptable carrier" as used herein should be taken to mean any inert, non-toxic, solid or liquid filler, diluent or encapsulating material, or other excipient, which does not react adversely with the active ingredients) or with a patient.
Such formulations and carriers are well known in the art and include pharmaceutical formulations that may be, for example, administered to a patient systemically, such as parenterally, or orally or topically.
The term 'parenteral' as used here includes subcutaneous, intravenous, intramuscular, infra-arterial and infra-tracheal injection, and infusion techniques.
Parenteral formulations are preferably administered intravenously, either in bolus form or as a constant infusion, or subcutaneously, according to known procedures. Preferred liquid carriers, which are well known for parenteral use, include sterile water, saline, aqueous dextrose, sugar solutions, ethanol, glycols and oils.
Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants and wetting agents, etc. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs or the like, or may be presented as a dry product for reconstitution with water or other suitable vehicle for use. Such liquid preparations may contain conventional additives, such as suspending agents, emulsifying agents, non-aqueous vehicles and preservatives.

Formulations suitable for topical application may be in the form of aqueous or oily suspensions, solutions, emulsions, gels or, preferably, emulsion ointments.
Unit doses of the pharmaceutical formulations according to the invention may contain daily required amounts of the haemostasins, or sub-multiples thereof to make a desired dose. The optimum therapeutically-acceptable dosage and dose rate for a given patient (which may be a mammal, such as a human) depend on a variety of factors, such as the potency of the active ingredient(s); the age, body weight, general health, sex and diet of the patient; the time and route of administration; rate of clearance; the object of the treatment (eg treatment or prophylaxis); and the nature of the disease to be treated.
It is expected that a systemic dose in the range of from~0.005 to 50 mg/kg body weight, preferably between 0.05 and 10 mg/kg and more preferably from 0.1 to 1 mg/kg, will be effective. According to the nature of the disease being treated, one single dose may contain in the range of from 0.05 to 10 mg/kg body weight, whether applied systemically or topically. Oral formulations are preferably administered in 2 to 6, preferably 3 to 4, sub-doses per day.
A further aspect of this invention provides covalent complexes of the haemostasins with the surfaces of prostheses or extracorporeal circulations that are exposed to blood in order to prevent either the initiation of complement and/or intrinsic coagulation and its pathological sequellae.
Because of their biological activity, described hereinabove and amplified in the following Examples, the present invention provides the use of a polypeptide, particularly a haemostasin, described herein or a pharmaceutical formulation thereof or a nucleic acid sequence coding therefor in the treatment or prophylaxis of a condition or disorder related to C 1 esterase initiation and/or factor 3~I activation. Alternatively, the present invention provides a method for the treatment or prophylaxis of a condition or disorder related to C 1 esterase initiation and/or factor XII activation, which method comprises the administration to a patient in need thereof of an effective, inhibitory amount of the polypeptide or a pharmaceutical formulation thereof. Preferably, the use or method is one wherein the medical condition or disorder is selected from one or more of cardiovascular disease, inflammation and auto-immune diseases.
For example, the haemostasins can potentially be used to inhibit the activation of coagulation, which happens to the detriment of patients in, for example, thrombotic disease selected from deep venous thrombosis, pulmonary embolus, and thrombosis associated with angioplasty and endarterectomy. Further, disease may also be alleviated by the ability of the haemostasins to inhibit both complement activation and the intrinsic pathway of blood coagulation, such as in haemodialysis, cardiopulmonary bypass, or rejection of transplanted organs or tissues, or in the syndromes: sepsis;
myocardial.
infarction; stroke; particularly in the injury caused to tissues by reperfusion after an ischaemic period (such as occurs after heart attack or cerebral stroke);
atherosclerosis;
shock; vasculitis; rheumatoid arthritis; sickle cell anaemia or angioedema. In addition, the haemostasins may be used in conditions associated with activation of complement as adjudged by the appearance of activated components or their complexes with natural inhibitors in biological fluids and/or their deposition in diseased tissues, such as: various autoimmune diseases (eg lupus arthritis); glomerulonephrids; nephritis;
nephropathy;
systemic sclerosis; Beh~ets syndrome; cerebral lupus; Guillan-Barre disease;
multiple sclerosis; myasthenia gravis; pemphigus; bullous pemphigoid; phototoxic reactions;
thermal burns; anaphylaxis; asthma; skin reactions; infections; inflammatory bowel disease; thyroiditis; infertility; Alzheimer's disease; paroxysmal nocturnal haemoglobinuria; and haemolytic anaemia.
A haemostasin of the invention or formulation thereof may advantageously be administered in combination with an additional anticoagulant or a thrombolytic agent to reduce tissue injury following reperfusion or to prevent complement activation on the surface of haemodialystr or cardiopulmonary bypass apparatus. Furthermore, haemostasins may be used in combination with immunosuppressant agents to decrease transplant rejection or with steroidal or non-steroidal anti-inflammatory drugs.
By the teen "in combination" is meant the simultaneous or sequential administration of the haemostasins with the other, pharmacologically active, ingredient(s).
Processes for the isolation and characterisation of the polypeptides according to the invention will now be described with reference to the accompanying sequence listings, by way of example only, in which:
52314 refers to H-D-Val-Sei Arg-4-nitroanilide;
S2266 refers to H-D-Val-Leu-Arg-4-nitroanilide;
52302 refers to H-D-Pro-Phe-Arg-4-nitroanilide;
S2366 refers to pyroGlu-Pm-Arg-4-nitroanilide; and S2238 refers to H-D-Phe-Pip-Arg-4-nitroanilide.

WO 99/3b439 PCT/GB99/00147 is EXAMPLE 1:
Inhibition of Classical Complement Pathway by Haementeria Extracts The occurrence of inhibitors of the classical complement pathway in leeches can be demonstrated by an immunoassay of the membrane attack complex. Extracts of different leech species were prepared by homogenisation of either the whole salivary complex or the individual glands in CAE (Diasorin Ltd, Charles House, Toutley Road, Wokingham, Berks, UK) diluent and assayed in the Diasorin CAE test. Samples containing CAE
diluent (0.5 mI), extract (0.095 ml) and normal human serum (0.005 ml) were made up and 0.15 ml aliquots placed in the antibody-coated wells. After incubation for 1 h at 37 °C, the sample was discarded and the wells were washed 3 times with wash buffer.
Bound membrane attack complex was probed with a specific antibody conjugated with horseradish peroxidase (0.15 ml) by incubation at 37 °C for 30 min.
Unbound antibody was removed by washing three times as above and the bound antibody was visualised with chromogen plus substrate solution (0.15 ml) and incubation for 30 min at room temperature. The reaction was stopped with stop solution. The contents of the well were transferred to a clean microtitre plate and read at 450 nm. The number of anti-CAE units was calculated on the basis that the control serum contained 100 CAE
units/well and 1 inhibitory unit inhibits 1 CAE unit by 100 %. The inhibitory activity is demonstrable in the salivary complexes of both Haementeria o~cinalis and H. ghilianii and is highest in the salivary glands (Table 1).
Table 1:
t Tissue Inhibitory units / leech Haementeria officinalisWhole salivary complex44,000 Haementeria ghilianiiWhole salivary complex1 175 000 , , Haementeria ghilianiiPosterior salivary 42,300 glands Haementeria ghilianiiAnterior salivary 63 300 glands , Haementeria ghilianiiProboscis 17,500 EXAMPLE 2:
Inhibition of Cls Component of Complement by Haemeaterla Extracts The presence of inhibitors of the Cls component of complement can be demonstrated by the ability of leech extracts to inhibit a chromogenic assay of the enzyme. An extract of a single whole salivary complex of Haementeria o,~icinalis, comprising anterior, posterior glands and proboscis;~was prepared by homogenisation in phosphate buffered saline (0.45 ml). An extract of the anterior salivary glands of a single Haementeria ghilianii was prepared by homogenisation in phosphate buffered saline (0.5 ml). Both extracts were centrifuged at 13,000 rpm for 3 min and the supernatant was used for the assay. Cuvettes were made up as follows: 8 mM S 2314 (0.01 ml); extract or phosphate buffered saline (0.04 ml); and O.1M sodium phosphate buffer pH 7.3 (0.14 ml). The reaction was started by addition of 0.025 rng/mi Cls (Calbiochem)(0.01 ml) and monitored in a Beckman DU
650 spectrophotometer by the increase in absorbance at 405 nm.
Table 2 shows the rate of the enzyme-catalysed reaction after subtraction of the rate of a similar cuvette where water replaced the enzyme. The reaction rate where water replaced enzyme was between 0.03 and 0.11 mAbs/min in each experiment. Table 2 demonstrates the ability of the extracts of both Haementeria o~cinalis and Haementeria ghilianii to inhibit C 1 s and indicates the presence of an inhibitor of this enzyme.
Table 2:
Extract mAbs/min Percent Inhibition Phosphate buffered 8.77 -saline Haementeria ghilianii -0.064 100 Haementeria o,~cinalis0.567 93.5 EXAMPLE 3:
Inhibition of Classical Complement Pathway by Active Fractions of H, ghilianii Owing to the abundance of the complement inhibitory factor in the posterior salivary glands of Haernenteria ghilianii, these were used as a source of material in this example.

WO 99/36439 PCTIGB99/~147 m Glands from 150 individual animals were homogenised in 20 tnM Tris HCl pH 8.0 (4 ml) and centrifuged. The pellet was extracted twice more with 4 ml aliquots of buffer and the supernatants were pooled. The supernatants were run on a 60 x 100 mm column of Q-Sepharose which had been equilibrated in 20 mM Tris HCl pH 8Ø Proteins were eluted with a linear gradient from the starting buffer to Tris containing 1M NaCI and detected by absorbance at 280 tnm. All fractions were dialysed against 10 mM Tris HCl pH
7.0 (20 voltunes) for two changes and then against water (20 volumes) before assaying in the CAE method (cf. Example 1 ). The inhibitory activity eluted at approximately 0.75 M
NaCI.
Further purification was achieved by chromatography on a 50 x 100 mm column of CM-Sepharose which had been equilibrated in 20 mM sodium acetate pH 5Ø The active fraction from the Q-Sepharose was dialysed against 20 mM sodium acetate pH 5.0 and applied to the cohunn. Proteins were eluted with a linear gradient from the starting buffer to the same buffer containing 1 M NaCI. Fractions were dialysed exhaustively against water before assay in the CAE method. The inhibitory activity eluted at approximately 0.6 M NaCI.
After lyophilisation, the active fraction was reconstituted in water (2 ml) and applied to a 16 x 600 mm column of Superdex-75 which had been equilibrated in phosphate-buffered saline pH 7.4. Direct assay of the fractions in the CAE method demonstrated that the inhibitory activity eluted in the major peak in 0.52-0.66 column volumes. The active fraction was homogeneous by SDS PAGE under reducing conditions and had an apparent molecular weight of 24.7 kDa.
A sample of the active fraction was run on a 5 x 100 mm reverse phase ProRPC
HPLC
column (Amersham Pharmacia Biotech, UK) which had been equilibrated in 0.1 %
v/v trifluoroacetic acid and eluted with a linear gradient from starting buffer to 75 % v/v acetonitrile in 0.1 % v/v trifluoroacetic acid. The elution was monitored by absorbance at 210 nm. A single peak was eluted which was homogeneous on SDS PAGE under reducing conditions and contained complement inhibitory activity as meastued by the CAE assay.

is EXAMPLE 4: Partial (N-Terminus) Amino Actd Sequence The fraction containing the peak from ProRPC HPLC described in example 3 contained a protein (hereinafter designated "haemostasin 1'~ which gave a single amino acid sequence from the N-terminus on an Applied Biosystems 473A automatic sequencer. After identifying the first 10 amino acids, the sequence of the first 29 amino acids was found:

X-K K-K-L-P-K-X'-Q-K-Q-E-D-C-G-S-W-D-L-K-C-N-W-V-T-K-K-C-E-wherein X and X' are as hereinbefore defined, respectively, [SEQ ID Nos: 14 and 15].
By digestion of the fiaction containing the peak from ProRPC HPLC with LysC
endoprotease and purification of the proteolytic fragments by ProRPC HPLC, two internal sequences of the polypeptide were identified.
This sequence information allowed the design of primers to enable production of double-stranded DNA for sequencing, according to Example 5.
EXAMPLE 5: mRNA and Partial cDNA Sequence from H. ghil~anu, Recombinant DNA Vectors and Plasmlds mRNA was extracted from the complete salivary complexes of 20 Haementaria ghilianii by thawing the finzen tissue in guanidinium thiocyanate lysis solution (8 ml) as described in Ambion Micro (A) Pure kit (Ambion Inc., 2130 Woodward Street, Austin, Texas, USA). Following Dounce homogenisation, dilution buffer (16 ml) was added, mixed and supernatant collected following centrifugation (12000 g, 4°C, 15 min).
The supernatant was mixed with oligo dT (deoxythymidine) resin (20 mg) at room temperature for 60 min and collected by centrifugation (4000 g, room temp, 3 min). The oligo dT
pellet was then treated to three cycles of addition of high salt binding buffer (1 ml), vortexing and centrifugation (4000 g, room temp, 3 min). The final pellet was re-suspended in wash buffer (0.5 mls) added to a spin column and centrifuged (5000 g, mom temp, 20 sec).
The column was washed three more times in the same manner by addition of wash buffer (Smls) and centrifugation. The bound Poly A+ RNA was finally collected by adding pre-warmed (65°C) elution buffer (100 pl), centrifugation and precipitation in the presence of ammonium acetate (20 ~tl), glycogen (1 ~1) and ethanol (550 ~1) and stored at -20°C.
Using half the mRNA isolated above, a complementary DNA strand was constructed by reverse transcription. The ethanolic preparation (400 pl) was centrifuged and the pellet dissolved in sterile water {8 pl). A reverse transcription reaction (RT-PCR) was set up as follows, using an Ambion Retroscript kit comprising: mRNA (7 pl), dNTPs mix (Boehringer Mannheim){2.5 mM each)(4 pl), random hexamer primers (2 ~1) and sterile water (3 pl). This solution was incubated at 85 °C for 3 min and the following additions made on ice: RT-PCR buffer (10 x concentrated, 2 ~1), placental RNAase inhibitor (1 ~1), M-MLV reverse transcriptase ( 1 ~,1). The reaction was allowed to proceed at 42 °C for 80 min and inactivated by incubation at 92°C for 10 min. The reaction was then stored at -20 °C.
RT-PCR was used to produce double-stranded cDNA for sequencing. Using the first strand cDNA prepared above and degenerate primers A and B corresponding to the known amino acid sequences from example 4, the following reaction was set up (Ambion Retroscript kit): reverse transcriptase reaction above (5 pl), reaction buffer (10 x concentrated, 5 pl), dNTPs mix (2.5 mM each) (2.5 ~1), 5 E,~M primers A (2.5 wl), 5 ~M
primers B (2.5 ~,1), Super Taq+, 2 units (Ambion)(0.5 pl) and sterile water {33 pl). The reaction mixture was denaxured at 94 °C for 2 min and cycled in a Techne Genius DNA
Thermal Cycler for 30 cycles each of 94 °C for 30 sec, 45 °C for 60 sec and 72 °C for 90 sec. The final incubation was at 72 °C for 10 min. The resulting PCR
sample was subjected to another round of PCR using different primers based on the amino acid sequences described in example 4 to increase specificity. The reaction mixture comprised: PCR Reaction above (5 wl), Taq Buffer {Life Technologies Ltd, Inchinnan Business Park, Paisley, UK)(10 x concentrated, 5 p,l, dNTPs mix (2.5 mM
each,)(2.5 ~,1), primers C (5(2.5 pl), primers D (5 pM)(2.5 ~1), Taq (Life Tec)(lU)(0.25 ~,1) and sterile water (34.75 pl). The reaction mixture was denatured at 94 °C
for 2 min and cycled 30 times in a Techne Genius DNA Thermal Cycler, as follows: 94°C
for 30 sec, 50 °C for 30 sec and 72 °C for 9U. sec. The final incubation was 72 °C for 10 min.
A single DNA band of approximately 250 base pairs was identified in the PCR
reaction when visualised by 1 % agarose gel electrophoresis. This DNA fi~agrnent was purified 5 using a Promega Wizard prep column and ligated to plasmid vector pCR2.1 (invitrogen TA Cloning kit). The resultant recombinant plasmids were introduced into competent E.
toll (INV aF') and stocks of recombinant clones and plasmid DNA generated.
Plasmids were sequenced on an ABI Sequencer and the cDNA sequence corresponding to the available amino acid sequence identified [SEQ ID No: 6].
EXAMPLE 6: Full cDNA Sequence Using the remaining half of the mRNA isolated in example 5, a new batch of cDNA was prepared and the Rapid Amplification of cDNA Ends (RACE) used to isolate the 5' and 3' ends of the whole sequence, as follows: The ethanolic solution of mRNA (400 pl) was centrifuged and the resulting pellet dissolved in sterile water (4 p,l). The reverse transcription reaction was set up as follows using a Clontech cDNA
amplification kit (Clontech Laboratories (LTK) Ltd., Wade Road, Basingstoke, UK): mRNA (4 ~1) and oligo dT primer ( 1 p,l). This solution was incubated at 70°C for 2 min and the following additions made on ice: first strand buffer (10 x concentrated, 2 ~1), dNTPs mix (10 mM
each)(1 ~,1), AMV reverse transcriptase (20 U)(1 p,l) and sterile water (1~1).
The reaction was allowed to proceed at 42°C for 60 min. The following additions were made on ice:
first strand reaction (10 ~,1), sterile water (48.4 ~1), second strand buffer (5 x concentrated, 16 ~l), dNTPs mix (10 mM each)(1.6 ~1) and 20 x second strand enzyme cocktail (4 ~1).
The mixture was incubated at 16°C for 90 min. This was followed by the addition of T4 DNA polymcrase (2 ~1) and incubation was continued for a further 45 min at 16°C. The reaction was terminated by the addition of 10 mM EDTA, and pure DNA extracted by phenol/chlomform and precipitated by ethanol. The pellet was dissolved in sterile water (10 ~.1).
Marathon adaptor sequence was ligated to the ends of the double stranded cDNA
to facilitate isolation, by incubation of double stranded cDNA (5 ~1), 5 x ligation buffer it (2 ~1), Marathon adaptor sequence ( 10 ~(2 p,l) and T4 ligase ( 1 L>)( 1 pl) for 14 hours at 16 °C and terminated at 70 °C for 5 min. This was followed by the addition of Tricine-EDTA buffer ( l OmM Tricirie-KOH pH 8.5, 0.1 mM EDTA)(240 ~1}.
Based on the sequence generated in example 5, specific primers (primers E and F) were designed. RACE was used to isolate the 5' and 3' ends of the whole sequence.
The following incubations were designed: 10 x PCR reaction buffer (5 ~1), dNTP mix (10 mM)(1 ml), 50 mM MgCl2 (1.5 pl), Taq (2Ln (0.5 ~1), sterile water (35 p,l) and for the 5' end as follows: cDNA (5 pl), AP1 Primer (Clontech)(1 p,l), 10 N,M primer E
[AGTGTTGCAAGTACAGAA] ( 1 ~1); or for the 3' end, as follows: cDNA (5 p,l), API
Primer (1 p.l), gene-specific primer F [AAGAAATGCGAATGCAGG](10 pM). The reaction mixtures were denatured at 94 °C for 2 min and cycled in a Techne Genius DNA
Thermal Cycler for 30 cycles of 94 °C for 30 sec, 45 °C for 60 sec and 72 °C for 90 sec with a final incubation at 72 °C for 10 min.
DNA fragments were purified as in example 5, sequenced and sequences corresponding to the amino acid sequence defined in example 4 identified. This allowed elucidation of two further nucleotide sequences coding for haemostasins: the complete DNA
sequence [SEQ
ID No: 4] and another variant DNA sequence [SEQ ID No: 5]. The complete amino acid sequences of three haemostasin variants [SEQ m Nos: 3, 7 and 8] were deduced from the DNA. The calculated molecular weight based on the amino acid sequence is approximately 14,200 Da, assuming no post-transiational modification. The nucleotide sequences [SEQ m Nos: 5 and 6] both contained code at the 5' end for a leader amino acid sequence [M S F K I V L L L F L V V C V V A S L A], which is not attached to the N-terminus of the extracted native protein.
EXAMPLE 7: Inhibition of Factor XII Activation by Haemostasin 1 The effect of haemostasin 1 (cf. Examples 3 and 4) on the activation of factor XII was investigated in a chromogenic assay. The cuvette contained: the supernatant from acetone-treated plasma (0.025 ml) prepared by treatment of human plasma with 0.33 volumes of acetone and incubation for 10 min; 50 mM Tris HCl containing 3.36 g/1 EDTA pH 7.9 (0.075 ml); 10_ pM soybean trypsin inhibitor (0.04 ml); inhibitor sample or phosphate-buffered saline (0:1 ml); and 40 ug/ml dextran sulphate (0.1 ml).
These were incubated at 20 °C for 10 min and the resultant factor /3XIIa was assayed after addition of 0.87 mM S2302 (Quadratech) in 50 mM Tris HCl pH 7.9, by measurement of the rate of absorbance change at 405 nm. Haemostasin 1 inhibited the activation of factor 3~I in a concentration-dependent manner. By varying the concentration of dextran sulphate, apparent competitive kinetics were demonstrable and the Ki was calculated to be 0.31 pg/ml.
EXAMPLE 8: Activated Partial Thromboplastin Assay - Haemostasin 1 The effect of haemostasin 1 was investigated on plasma clotting by standard methods.
Human plasma samples were made up to contain a range of haemostasin 1 concentrations from 0.02 to 1.23 pM or with the equivalent buffer controls and the time for clotting to occur when activated by tissue thromboplastin (one stage prothrombin time) or thrombin (thrombin time) or thrombosil (Ortho Diagnostics, Amersham, Bucks.) (activated thromboplastin time) was measured on a Sysmex CA5000 automated coagulometer.
There was no effect on the one stage prothrombin time, a measure of the extrinsic coagulation pathway, and no effect on the thrombin time. However haemostasin 1 prolonged the activated partial thromboplastin time. A concentration of 3.8 ~,g/ml prolonged the activated thromboplastin time by 50%. The effect of haemostasin 1 on factor XIIa is therefore reflected in its ability to inhibit the clotting of plasma.
EXAMPLE 9: Dose-Responsive Effect of Haemostasin 1 in CAE Assay The ICso of haemostasin 1 prepared as in example 3 was investigated in the CAE
assay as described in example 1. By varying the concentration of haemostasin in the assay, the effect was shown to be dose-responsive with an ICso of 0.241 pg/ml, EXAMPLE 10: Classical C$,° Assay _ ~Isemostasin 1 Inhibitors of the classical complement pathway can be demonstrated in a haemolytic assay whereby they inhibit the ability of complement to lyse antibody-coated erythrocytes. Lysis is quantified by measuring the released haemoglobin at 405 nm. The ICso of haemostasin 1 prepared as in example 3 was investigated in a standard haemolytic (CH5°) assay. Sheep erythrocytes were washed 3 times in triethanolamine-buffered saline (TBS-G) (128.3 mM NaCI, 17.7 mM HCI, 20.6 mM triethanolamine, 0.5 mM MgCI,, 0.15 mM CaCh, 0.05 % (w/v) gelatin, pH 7.35) by centrifugation at 2000 rpm for 10 min and re-suspension. The erythrocytes were coated with antibody by adding haemolysin (Harlan-SeraLab), diluted 1:200 in TBS-G (4 ml) to erythrocytes (4 ml, diluted 1:4 in TBS-G) and incubating at 37°C for 30 min then at 0°C for a further 30 min with periodic mixing. The coated erythrocytes were washed twice in TBS-G and re-suspended in TBS-G supplemented with 2.5 % (w/v) glucose and 0.1 % (w/v) sodium azide, and diluted in TBS-G to give an absorbance at 405 nm of 0.7 when fully lysed.
For the assay, blank microtitre plate wells contained TBS-G (0.2 ml) or water plus coated erythrocytes (0.05 ml) giving absorbance values for 0 % and 100 % haemolysis, respectively. Test wells contained TBS-G (0.1 ml), haemostasin or phosphate-buffered saline (PBS) (0.05 mi), human serum (complement) diluted in TBS-G to give approximately 75 % haemolysis (0.05 ml) and erythrocytes (0.05 ml). The plate was covered and incubated at 37°C for 1 h and centrifuged at 1000 rpm for 3 min.
Supernatants (0.2 ml) were transferred to a flat well microtitre plate and absorbance was read at 405 nm. The percent haemolysis was compared with the PBS control (Table 3).
By varying the concentration of haemostasin in the assay, the effect was shown to be dose-responsive with an ICs° of 0.019 pg/ml.
Table 3:
[Haemostasin 1 ](ng/ml) % haemolysis EXAMPLE I1: Alternative Complement Pathway - No Inhibition by Haemostasin 1 In addition to the classical route, complement can also be activated by the alternative pathway by chelation of calcium ions and provision of magnesium ions [Servais G, Walmagh J, Duchateau J. J htimunol Meth 140:93-100 (1991)]. Rabbit erythrocytes were washed 4 times in gelatin veronal buffer (Sigma) containing 10 mM EGTA and 7 mM
MgCh pH 7.2 (ie VCM-MEG) by centrifugation at 2000 rpm for 10 min and re-suspension. Microtitre plate wells contained VCM-MEG (0.15 ml), test sample (0.075 ml) human plasma diluted 1:4 with VCM-MEG (0.075 ml), 1 % vlv washed erythrocytes (0.075 ml). The plate was covered and incubated at 37 °C for 45 min before addition of 0.2 M EDTA (0.0225 ml), centrifugation at 1000 rpm for 3 min and transfer of the supernatants (0.1 ml) to a fresh flat-bottomed microtitre plate. Absorbance was read at 405 nm. The percent haemolysis was compared with a PBS buffer blank, samples where the plasma was omitted (0 % haemolysis) or where water (0.015 ml) was substituted for the VCM-MEG buffer (100 % haemolysis). There was no significant inhibition of the alternative pathway by haemostasin 1 at concentrations up to 20 p,g/ml.
EXAMPLE 12: Selectivity of Ilaemostasin 1 for Complement Cls The selectivity of haemostasin 1 for various serine proteases was determined in chromogenic substrate assays. Assays were set up using commercially-available substrates at concentrations at or near to the published Km. Enzyme rate curves were monitored by the release of 4-nitroanilide from the substrate at 405 nm in a spectrophotometer in cuvettes containing various concentrations of haemostasin 1. Tissue kallila~ein was Padutin (Bayer AG, Leverkusen, Germany). Plasma kallila~ein was obtained from Quadratech, (Epsom, Surrey, UK).
Factor (3XIIa was activated from normal human plasma by treatment of the plasma with 0.33 volumes of acetone, followed by centrifugation. The acetone-treated plasma (0.025 ml), 50 mM Tris HCl containing 3.36 g/1 EDTA pH 7.9 (0.075 ml), phosphate buffered saline (0.1 ml), 10 pNi soy bean trypsin inhibitor (Sigma)(0.04 ml) and 40 ~glml dextran sulphate (0.1 ml) were incubated for 10 min at 37 °C prior to addition of 0.87 mM
substrate S2302 in 22 mM Tris HCl pH 7.9 (0.46 ml) in the presence of either haemostasin 1 or its vehicle', phosphate buffered saline (0.1 ml).
5 Factor XIa was activated similarly from normal human plasma and the assays were carried out with S2366 in the presence of 2.5 pg/ml corn trypsin inhibitor (Rho reagents, Gerrards Cross, Bucks, UK) to inhibit substrate cleavage by factor (3XIIa.
Human thrombin was obtained from NIBSC (Blanche Lane, Potters Bar, UK). The 10 enzymes, Clr (Sigma) and Cls (Calbiochem), were incubated at 37 °C
for 1 h prior to use to allow their activation. Complement factor D was purchased from Sigma Chemical Company.
Table 4 shows that haemostasin 1 only inhibited complement C 1 s of all of these enzymes, 15 having no effect at concentrations up to 7.4 pg/ml on the others.
Table 4:
Enzyme Enzyme Substrate ICso (wg/ml) Concentration Tissue kallikrein0.15 KU/ml 0.05 mM S2266 X7,4 Plasma kallikrein0.53 mPEU/ml 0.2 mM 52302 X7,4 Factor (3XIIa 0.023 PEU/ml 0.5 mM S2302 X7,4 Factor XIa 0.023 PEU/ml 0.8 mM S2366 X7,4 Thrombin 0.006 unit/ml0.1 mM S2238 X11.7 Complement C 1.25 wg/ml S2314 0.043 1 s Complement C 10 pg/ml 0.8 mM S2314 X7.04 1 r Complement factor1.25 pg/ml 0.3 mM Na-CBZ-lysine-SBzIX7.4 D

~ = no effect at this concentration.
20 * = Ki determined by varying the concentration of both S2314 and haemostasin 1 and analysis by the Lineweaver-Burk method.

EXAMPLE 13: Inhibition of Factor XI Activation by Haemostaain 1 Since factor XI activation is dependent on the generation of factor aXIIa, it was expected that its activation would.also be inhibited by haemostasin 1. Plasma was activated in the presence or absence of different concentrations of haemostasin 1 in a glass cuvette containing acetone-treated plasma (0.025 ml), 50 mM Tris HCl containing 3.36 g/1 EDTA
pH 7.9 (0.075 ml), 10 ~M soybean trypsin inhibitor (0.04 ml), inhibitor sample or phosphate buffered saline (0.1 ml) incubated at 20 °C for i 0 min. A
0.3 ml sample was assayed for factor XIa activity by mixing in a cuvette containing 50 mM Tris HCl pH 7.9 (0.26 ml), 50 ~.g/ml corn trypsin inhibitor (0.04 ml) and 4 rnM S2366 (0.2 ml). Factor XI
activation by glass was inhibited by haemostasin 1 with an ICso of 7.4 pg/ml.

SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Bio-Discovery Ltd (B) STREET: Unit 15, Pontardulais Workshop, Tynybonau Road, Pontardulais (C) CITY: Swansea (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): SA4 1SG
(ii) TITLE OF INVENTION: Polypeptides, DNA encoding thereof, formulations thereof and their use in inhibiting factor XII
(iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: PCT/GB99/......

(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Xaa Lys Lys Lys Leu Pro Lys Xaa Gln Lys (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TXPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Lys Lys Lys Leu Pro Lys Cys Gln Lys Gln Glu Asp Cys Gly Ser SUBSTITUTE SHEET (RULE 26) WO 99!36439 PGT/GB99/00147 Trp Asp Leu Lys Cys Asn Asn Val Thr Lys Lys Cys Glu Cys Arg Asn Gln Val Cys Gly Arg Gly Cys Pro Lys Glu Arg Tyr Gln Arg Asp Lys Tyr Gly Cys Arg Lys Cys Leu Cys Lys Gly Cys Asp (2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
Ala Lys Lys Lys Leu Pro Lys Cys Gln Lys Gln Glu Asp Cys Gly Ser Trp Asp Leu Lys Cys Asn Asn Val Thr Lys Lys Cys Glu Cys Arg Asn Gln Val Cys Gly Arg Gly Cys Pro Lys Glu Arg Tyr Gln Arg Asp Lys Tyr Gly Cys Arg Lys Cys Leu Cys Lys Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys Thr Cys Asn Thr Lys Glu Thr Ala Cys Val Asn Ile Trp Cys Thr Asp Pro Tyr Lys Cys Asn Pro Glu Ser Gly Arg Cys Glu Asp Pro Asn Glu Glu Tyr Glu Tyr Asp Tyr Glu (2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 607 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:

SUBSTITUTE SHEET (RULE 26) CAGGAAGACT

AGGAATCAAG

TGCAGGAAGT

GGCTTTAAGA

GCATGTGTGA

GAAGACCCAA

ACCATACATC

ATTCTTGGTA

TTATTTTATT

(2) INFORMATION FOR SEQ ID N0: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 321 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:

(2) INFORMATION FOR SEQ ID N0: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:

SUBSTITUTE SKEET ~pULE Zg) (2) INFORMATION FOR SEQ ID N0: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122. amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
Ala Lys Lys Lys Leu Pro Lys Cys Gln Lys Gln Glu Asp Cys Gly Thr Trp Asp Leu Lys Cys Asn Asn Val Thr Lys Lys Cys Glu Cys Arg Asn Gln Val Cys Gly Arg Gly Cys Pro Lys Glu Arg Tyr Gln Arg Asp Lys Tyr Gly Cys Arg Lys Cys Leu Cys Lys Gly Cys Asp Gly Phe Lys Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys Thr Cys Asn Thr Lys Glu Thr Ala Cys Val Asn Ile Trp Cys Thr Asp Pro Tyr Lys Cys Asn Pro Glu Ser Gly Arg Cys Glu Asp Pro Asn Glu Glu Tyr Glu Tyr Asp Tyr Glu (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 8:
Ala Lys Lys Lys Leu Pro Lys Cys Gln Lys Gln Glu Asp Cys Gly Ser SUBSTITUTE SHEET (RULE 26) Trp Asp Leu Lys Cys Asn Asn Val Thr Lys Lys Cys Glu Cys Arg Asn Gln Val Cys Gly Arg Gly Cys Pro Lys Glu Arg Tyr Gln Arg Asp Lys Tyr Gly Cys Arg Lys Cys Leu Cys Lys Gly Cys Asn Gly Phe Lys Cys Arg Leu Gly Cys Thr Tyr Gly Phe Lys Thr Asp Lys Lys Gly Cys Glu Ala Phe Cys Thr Cys Asn Thr Lys Glu Thr Ala Cys Val Asn Ile Trp Cys Thr Asp Pro Tyr Lys Cys Asn Pro Glu Ser Gly Arg Cys Glu Asp Pro Asn Glu Glu Tyr Glu Tyr Asp Tyr Glu (2) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 9:
Xaa Lys Lys Lys Leu Pro Lys Cys Gln Lys (2) INFORMATION FOR SEQ ID N0: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Xaa Lys Lys Lys Leu Pro Lys Glu Gln Lys (2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids (8) TYPE: amino acid SUBSTITUTE SHEET (RULE 261 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Ala Lys Lys Lys Leu Pro Lys Cys Gln Lys (2) INFORMATION FOR SEQ ID N0: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Ala Lys Lys Lys Leu Pro Lys Glu Gln Lys (2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Ala Lys Lys Lys Leu Pro Lys Cys Gln Lys Gln Glu Asp Cys Gly Ser Trp Asp Leu Lys Cys Asn Asn Val Thr Lys Lys Cys Glu Cys Arg Asn Gln Val Cys Gly Arg (2) INFORMATION FOR SEQ ID N0: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SUBSTITUTE SHEET (RULE 26~

(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 14:
Xaa Lys Lys Lys Leu Pro Lys Cys Gln Lys Gln Glu Asp Cys Gly Ser Trp Asp Leu Lys Cys Asn Asn Val Thr Lys Lys Cys Glu (2) INFORMATION FOR SEQ ID N0: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear tii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Xaa Lys Lys Lys Leu Pro Lys Glu Gln Lys Gln Glu Asp Cys Gly Ser Trp Asp Leu Lys Cys Asn Asn Val Thr Lys Lys Cys Glu SUBSTITUTE SHEET (RULE 26)

Claims (31)

1. An isolated palypeptide obtainable from the salivary gland of a Haementeria leech, wherein the polypeptide has an electrophoretic mobility on SDS PAGE
consistent with an apparent molecular weight of from 20 to 25 KDa, and wherein the polypeptide can inhibit at least one of Cl esterase or factor XII
activation.

2. The isolated polypeptide of claim 1, wherein the Haementeria leech is either Haementeria ghilanii or Haementeria officialis.

3. The isolated polypeptide of claim 2, wherein the Haementeria leech is Haementeria ghilanii.

4. The isolated polypeptide of any one of claims 1 and 2, wherein the polypeptide comprises, at the N-terminus, the following sequence: X-K-K-K-L-P-K-X'-Q-K
(SEQ ID NO:1), wherein X is any natural amino acid, and X' is cysteine or glutamic acid.

5. The isolated polypeptide of claim 4, whsrein X is alanine.

6. The isolated polypoptide of claim 4, wherein the polypeptide comprises, at the N-terminus, the amino acid sequence of SEQ ID NO:14 or SEQ ID NO;15.

7. An isolated polypeptide, comprising amino acid residues 1 to 37 of SEQ ID
NO:3 (SEQ ID NO;13).

8. The isolated polypeptide of claim 7, further comprising amino acid residues to 60 of SEQ ID NO:3 (SEQ ID NO:2).

9. The isolated polypeptide of claim 8, comprising the amino acid sequence of SEQ ID NO:3.

10. The isolated polypeptide of any of claims 7, 8, or 9, further comprising a leader sequence, which resides at the N-terminus of the polypeptide.

11. An isolated polypeptide, comprising an amino acid sequence that is at least 80%
identical to the amino acid sequence of SEQ ID NO:3.

12. The isolated polypeptide of Claim 11, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:7.

13. The isolated polypeptide of claim 11, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:8.

14. An isolated nucleic acid molecule that encodes an amino acid sequence comprising the polypeptides of any one of claims 7, 8, 9, 12, or 13.

15. The isolated nucleic acid molecule of claim 14, comprising the nucleotide sequence of SEQ ID NO:5 or SEQ ID NO:6.

16. The isolated nucleic acid molecule of claim 14, wherein the nucleic acid molecule encodes an amino acid sequence that further comprises a leader sequence that resides at the N-terminus of the amino acid sequence.

17. The isolated nucleic acid molecule of claim 16, comprising the nucleotide sequence of SEQ ID NO;4.

18. The isolated nucleic acid molecule of any one of claims 14 to 17, wherein the nucleic acid molecule is DNA.

19. An isolated aucleic acid molecule that hybridises to a nucleotide sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID
NO:6, wherein the complement of the nucleic acid molecule encodes a polypeptide that can inhibit at least one of C1 esterase or factor XII
activation.

20. A vector, comprising a nucleic acid molecule that encodes an amino acid sequence comprising the polypeptides of any one of claims 7, 8, 9, 12, or 13.

21. A host that comprises the vector of claim 20.

22. The host of claim 21, wherein the host is a cell or a virus.

23. The use of a polypeptide according to any one of claim 1 to 13 in the manufacture of a medicament.

24. A pharmaceutical formulation comprising a pharmaceutically acceptable carrier, and a polypeptide according to any one of claims 1 to 13.

25. A pharmaceutical formulation according to claim 24 for parenteral, oral, or topical use.

26. The use of a polypeptide according to any one of claims 1 to 13, to inhibit at least one of C1 esterase and factor XII activation.

27. The use of claim 26, wherein the palypeptide is administered to a subject having at least one condition or disease selected from the group consisting of cardiovascular disease, inflammation, and auto-immune disease.

28. A method for the treatment or prophylaxis of a condition caused by Cl esterase initiation or factor XII activation, wherein the method comprises the administration to a patient in need thereof of an effective, inhibitory amount of a golypeptide according to any one of claims 1 to 13, or a pharmaceutical formulation thereof.

29. A method according to claim 28, wherein the method comprises the administration of a composition comprising a nucleic acid molecule that encodes a polypeptide according to any one of claims 1 to 13.

30. The method according to any one of claims 28 and 29, wherein the polypeptide or nucleic acid molecule is administered to a subject having at least one condition or disease selected from the group consisting of cardiovascular disease, inflammation, and auto-immune disease,

31. A method for the preparation of a polypeptide according to any one of claims 1 to 13, wherein the method comprises either (a) isolating tissue or purifying extracts from a Haementeria leach, or (b) expressing a nucleic acid molecule encoding the polypeptide, and, optionally, isolating or purifying the resulting potypeptide.