CZ164494A3 - Human variant of kunitz-type protease inhibitor - Google Patents

Abstract

A variant of human Kunitz-type protease inhibitor domain I of tissue factor pathway inhibitor (TFPI), the variant comprising the following amino acid sequence X<1> Cys Ala Phe Lys Ala Asp X<2> Gly X<3> Cys X<4> X<5> X<6> X<7> X<8> X<9> Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe X<10> Tyr Gly Gly Cys X<11> X<12> X<13> Gln Asn Arg Phe X<14> Ser Leu Glu Glu Cys X<15> X<16> Met Cys Thr Arg X<17> (SEQ ID No. 1), wherein X<1> represents H or 1-7 naturally occurring amino acid residues except Cys, X<2>-X<16> each independently represents a naturally occurring amino acid residue, and X<17> represents OH or 1-5 naturally occurring amino acid residues except Cys, with the proviso that at least one of the amino acid residues X<1>-X<17> is different from the corresponding amino acid residue of the native sequence.

Description

Technical field

The present invention relates to a variant of the human domain of a Kunitz-type protease inhibitor, the DNA encoding the variant, a process for producing the variant, and a pharmaceutical composition comprising the variant.

BACKGROUND OF THE INVENTION

Polymorphonuclear leukocytes (neutrophils or PMN) and mononuclear phagocytes (monocytes) play an important role in tissue injury, infection, acute and chronic inflammation and wound healing. These cells migrate from the blood to the site of inflammation and, upon appropriate stimulation, release oxidizing compounds (O / O / H ₂ O ₂ and HOCl) as well as? granules containing a variety of proteolytic enzymes. The secreted granules include, but are not limited to, alkaline phosphatase, metalloproteinases such as gelatinase and collagenase, and serine proteases such as neutrophil elastase, cathepsin G and proteinase-3.

Latent metalloproteinases are released together with a tissue metalloproteinase inhibitor (TIMP). The mechanism of activation is not yet fully understood, but it is likely that oxidation of thiol groups and / or proteolysis plays a role in this process. Also ?

free metalloproteinase activity is dependent on TIMP inactivation.

In azurophilic leukocyte granules, serine protease neutrophil elastase, cathepsin G and proteinase-3 exist as active enzymes complexed with glucosaminoglycans. These complexes are not active, but dissociate and release active enzymes upon secretion. To neutralize the protease activity, large amounts of plasma inhibitor-proteinase inhibitor ATJ (a _x -PI) and .alpha.-chymotrypsin inhibitor, and _x (ATJ-Chl). However, PMNs are able to inactivate inhibitors locally. Thus, PI, which is the most important neutrophil elastase inhibitor, is sensitive to oxidation in the reaction center (Met-358) of oxygen metabolites produced by activated PMN. This reduces by approximately 2000 times the affinity and _x PI for neutrophil elastase.

After local neutralization and _x- PI, elastase is able to degrade numerous inhibitors of other proteolytic enzymes. Elastase cleaves and _x -ChI and thus promotes cathepsin G activity. It also cleaves TIMP, resulting in tissue degradation by metalloproteinases. Furthermore, the elastase cleaves antithrombin III, heparin cofactor II, and tissue factor pathway inhibitor (TFPI), which may promote clot formation. On the other hand, the ability of the neutrophil elastase to degrade coagulation factors is believed to have the opposite effect, so that the overall effect of the elastase is not clear. The effect of neutrophil elastase on fibrinolysis is less ambiguous. Fibrinolytic activity increases when the elastase cleaves the plasminogen activator inhibitor and the α- ₂ -plasmin inhibitor. - addition - of both - inhibiiory_g .These-sou "oxidized and _de- _t. activated metabolites 0 ₂ .

PMNs contain large amounts of serine proteases. About 200 mg of each leukocyte protease is released daily to act on invasive agents in the body. Acute inflammation leads to a multiple increase in the amount of enzyme released. Under normal conditions, proteolysis is maintained at an acceptably low level by large amounts. -PI inhibitor and _x and _x AA -CHI ₂ macroglobulin. However, there are some indications that many chronic diseases are caused by pathological proteolysis due to PMN over-stimulation, caused for example by autoimmune response, chronic infection, tobacco smoke or other irritants, etc.

Aprotinin (a bovine pancreatic trypsin inhibitor) is known to inhibit various serine proteases, including trypsin, chymotrypsin, plasmin and kallikrein, and to be used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic haemorrhage and heart attack (see, for example, JE Trapnell et al., Brit. J. Surg. 61, 177 (1974), J. McMichan et al., Circulatory Shock 9, 107 (1982),

L. M. Auer et al., Acta Neurochir. 49, 207 (1979);

Am, J. Obstet. Gynecol. 129, 164 (1977) and B. Schneider, Artzneim-Forsch. 26, 1606 (1976). Administration of aprotinin at high doses significantly reduces blood loss in cardiac surgery, including cardiopulmonary bypass surgery (see, for example, BP Bidstrup et al .: J. Thorac. Cardiovasc. Surg. 97, 364 (1989) and W. van Oeveren et al., Ann. Thorac Surg., 44, 640 (1987). It has previously been shown (see HR Wenzel and H. Tschesche: Angew. Chem. Dorm. Ed. 20, 295 (1981).) That some aprotinin analogs, eg aprotinin (1-58, Vall5), show a relatively high selectivity for granulocyte elastase and collagenase inhibitory effect, aprotinin (1-58, Ala1) has a weak influential elastase, while aprotinin (3-58, Arg15, Ala1, Ser42) shows an excellent inhibitory effect of plasma kallikrein (see WO 89/10374) .

However, when administered in vivo, α-protinine has been found to have a nephrotoxic effect on rats, rabbits and dogs after repeated injections with relatively high doses of aprotinin (Bayer: Trasylol, Inhibitor of Proteinase, E. Glaser et al., Verhandlungen). der Deutschen Gesellschaft fur Innere Medizin,

78. Kongress, Bergmann, pp. 1612-1614, Munich, 1972.)?

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The nephrotoxicity (manifesting inter alia as an injury) observed with aprotinin could be attributed to the accumulation of aprotinin in proximal tubular kidney cells as a result of the high positive charge of aprotinin that causes it to bind to negatively charged surfaces of these channels. This nephrotoxicity renders aprotinin less suitable for clinical purposes, especially those requiring administration of large doses of inhibitor (such as cardiopulmonary bypass surgery). In addition, aprotinin is a bovine protein, which may therefore contain one or more epitopes, which may cause an undesired immune response when administered to a human.

It is therefore an object of the present invention to identify human protease inhibitors of the same type as aprotinin (i.e. Kunitz type inhibitors) having a similar inhibitory profile or modified to exhibit the desired inhibitory profile.

SUMMARY OF THE INVENTION

The present invention relates to a human domain I variant of the Kunitz-type tissue factor inhibitor (TFPI) protease inhibitor. This variant comprises the following amino acid sequence

X ^X Cys Ala Phe Lys Ala Asp ^X2 Gly X ^with Cys X ⁴ X ⁶ X ⁶ X ⁷ X ⁸ X 'Phe Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe ^X10 Tyr Gly Gly Cys X ¹¹ x ^x? X ¹³ Gin Asn Arg Phe X ¹⁴ ser Leu Glu Glu Cys X ^xs X ^xs Met Cys Thr Arg X ¹⁷ (SEQ ID NO: 1) in which X ^x represents a hydrogen atom or 1 to 7 naturally occurring amino acid residues except Cys, X ² to X 1 are independently of each other a naturally occurring amino acid residue except Cys and X ¹⁷ is a hydroxyl group or 1 to 5

--- zbytkůpřirozeněsevyskvtujících amino acids except Cys, with the at least one of amino acid residues X ^X to X ¹⁷ is an amino acid residue other than the corresponding amino acid residue of the native sequence.

The term naturally occurring amino acid residue means the residue of any of the 20 naturally occurring amino acids, i.e., Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg and His.

TFPI, also known as an external pathway inhibitor (ÉPI) or a coagulation inhibitor associated with lipoprotein (LACI), was isolated by Broz et al. (Proc. Natl. Acad. Sci. USA et al., 1886

...............— (1997j-aeuropean..patent.300 ......, 9.8.8 ,.) .. The gene encoding this protein has been cloned, see European Patent 318 451. Analysis of the secondary structure of a protein shows that this protein has three Kunitz-type inhibitor domains, from amino acid 22 to amino acid 79 (I), from amino acid 93 to amino acid 150 (II), and from amino acid 185 to amino acid 242 (III). ). Kunitz type I domain TFPI has been shown to bind TF / FVIIa, while Kunitz type II domain II binds FXa (Girard et al., Nature 338, 518 (1989)).

By substituting one or more amino acids at one or more of the above-mentioned positions, it is possible to change the profile of the Kunitz-type TFPI domain inhibitor so that it preferentially inhibits neutrophil elastase, cathepsin G and / or proteinase-3. Further, it is possible to construct variants that specifically inhibit enzymes that are present in coagulation or fibrinolysis (eg, plasmin or plasma kallikrein), or a complement cascade.

One advantage of the Kunitz-type TFPI domain I is that it has a negative charge as opposed to aprotinin, which, as mentioned above, has a strong positive charge. Thus, it is possible to construct variants of the invention with a lower positive charge than aprotinin and thereby reduce the risk of kidney damage when administered at high doses of these variants. Another advantage is that, unlike aprotinin, it is a human protein (fragment), so that undesirable immunological reactions when administered to humans are significantly reduced.

Examples of preferred Kunitz-type TFPI domain I variants are those wherein X ¹ is Ser-Phe or Met-His-Ser-Phe, or wherein X ² is an amino acid residue selected from. a group consisting of Ala, Arg, Thr, Asp, Pro, Glu, Lys, Gln,

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Ser, Ile, and Val, especially those wherein X ³ is Thr or Asp, or wherein X ³ is an amino acid residue selected from the group consisting of Pro, Thr, Leu, Arg, Val, and Ile, especially those wherein X ³ is Pro or Ile, or wherein X ⁴ is an amino acid residue selected from the group consisting of Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Het, Trp, Tyr, Gln, Asn, and Ala, particularly such wherein X ⁴ is Lys, Val, Leu, Ile, Thr, Met, Gln or Arg, or wherein X ^s is an amino acid residue selected from the group consisting of Ala, Gly, Thr, Arg, Phe, Gln, and Asp, especially those in which X ⁵ is Ala, Thr, Asp or Gly, or wherein X ^fi is 2bytek amino acid selected from the group consisting of Arg, Ala, Lys, Leu, Gly, His, Ser, Asp, Gln, Glu, Val, Thr, Tyr, Phe, Asn, Ile and Met, in particular those in which X ⁶ is Arg, Phe, Ala, Ile, Leu or Tyr or where X ⁷ is an amino acid residue selected from the group of six dripping of Ile, Met, Gln, Glu, Thr, Leu, Val and Phe, particularly those wherein X ⁷ is Ile or where X® 2namená amino acid residue selected from the group consisting of Ile, Thr, Leu, Asn, Lys, Ser, Gln, Glu, Arg, Pro, and Phe, especially those wherein X ® is Ile or Lys, or wherein x ® is an amino acid residue selected from the group consisting of Arg, Ser, Ala, Gln, Lys, and Leu, especially those wherein X ⁹ is Arg, or wherein X ¹⁰ is an amino acid residue selected from the group consisting of Gln, Pro, Phe, Ile, Lys, Trp, Ala, Thr, Leu, Ser, Tyr, His, Asp, Met, Arg, and Val, especially those in which X ¹⁰ is Val or Ile, or wherein X ¹¹ is an amino acid residue selected from the group consisting of Gly, Met, Gln, Glu, Leu, Arg, Lys, Pro, and Asn, especially those in which X ¹¹ is Arg or Glu, or wherein X ¹² is Ala or Gly, or wherein X ¹³ is an amino acid residue selected from the group consisting of Ly s, Asn and Asp, especially those in which X ¹³ is Lys or Asn, or in —in-X ^{14 is} an amino acid residue / selected from the group consisting of Val, Tyr, Asp, Glu, Thr, Gly, Leu, Ser, Ile, Gin, His, Asn, Pro, Phe, Met, Ala, Arg, Trp and Lys, especially those wherein X ¹ * is Lys or Glu, or wherein X ¹⁵ is Lys, Met, Glu or Leu, or wherein X ¹⁶ is Lys, Ala, Asn or Glu, or wherein X ¹⁷ is Asp. In a preferred embodiment, X ¹ is Met-His-Ser-Phe and X ¹⁷ is Asp, at. wherein X ² to X ¹⁶ are as defined above.

Preferably, the Kunitz-type domain TFPI variants of the invention should preferably not contain a Met residue in the protease binding region (i.e., amino acid residues X ³ to X ¹⁴ ). Analogous to the above-described α1-PI, the Met residue at any of these positions would cause an inhibition of the oxidative deactivation of the oxygen metabolites produced by PMN and vice versa. , the lack of a Met residue at these positions would render the inhibitor more stable in the presence of these oxygen metabolites.

A commonly preferred variant of the invention is one in which one or more amino acid residues are located at the protease binding site of the Kunitz domain (i.e., one or more sites of X ³ and X ¹⁴ corresponding to positions 13, 15, 16, 17, 18, 19). ,

20, 34, 39, 40, 41 and 46 in aprotinin) substituted with amino acids present in the same positions of natural aprotinin.

This variant contains the following amino acid sequence:

Met His Ser Phy Cys Ala Phy Lys Ala Asp Asp Gly For Cys Lys Ala

Arg Ile Ile Arg Phe Phe Phe Asn Ile Phe Thr

Phe Val Tyr. Gly Gly Cys Arg. Ala Lys Gin Asn Arg. Phe Lys Ser Leu

Glu Glu Cys Lys Met Lys Met Cys Thr Arg Asp (SEQ ID NO: 2).

In another aspect, the invention relates to a DNA construct encoding a human variant of a Kunitz-type inhibitor domain of the invention. The DNA construct of the invention can be prepared synthetically by standard methods, e.g. the phosphoamidite method described by SL Beaucag et al. H. Caruthers: Tetrahedron Letters 22, 1859 (1981) or as described by Matthes et al., EMBO J. 2, 801 (1984). According to the phosphoamidite Me- * 'tody oligonucleotides are synthesized, e.g. in an automatic ^DNA? synthesizer, purified, annealed, ligated, and cloned in suitable vectors.

and

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It is possible to use genomic or cDNA coding TFPL ¹ Kunitztype domain I (e.g. obtained by screening a genomic or '. ^CDNA library for DNA coding for TFPI using synthetic oligonucleotide probes and isolating the DNA sequence that encodes Domain I). This DNA sequence is modified at one or more sites corresponding to the site (s) at which it is desired to make amino acid substitutions, eg, by site directed mutagenesis using synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination according to well known procedures.

In another aspect, the invention relates to a recombinant expression vector comprising a DNA construct of the invention. A recombinant expression vector can be any vector that can be suitably subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously repli8 kující vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., ^-. plasmid. The vector may also be a unit which upon introduction into the host cell integrates into the genome of the host cell and replicates together with the chromosome (s) into which it is integrated.

In the vector, the DNA sequence encoding the Kunitz-type tfpi variant I of the invention should be operably linked to a suitable promoter sequence. The promoter may be any DNA sequence that has transcriptional activity in the selected host cell. It may be derived from genes encoding proteins that are either homologous or heterologous to the host cell. her. Examples of suitable promoters for directing transcription of the DNA encoding the Kunitz-type TFPI variant of the invention of the present invention

Mammalian cells, the sau, ss40 promoter (Subramani et al., Mol. Cell Biol. 1, 854 (1981)), the MT-1 (metallothionein gene) protomer (Palmiter et al. Science 222 ₍ 809) (1983)) or adenovirus 2 major late promoter. Promoters suitable for use in yeast host cells include those from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255. 12073 (1980), Alber and Kawasaki: J. Mol. Appl. Gen. i, 419 ( 1982).) Or alcohol dehydrogenase genes (Young et al. In Genetic Engineering of Microorganisms for Chemicals, (Hollaender et al., Ed.), Plenum Press, New York, 1982.), TPI1 (U.S. Patent 4,599,311) or ADH2-4C (Russell et al., Nature 304. 652 (1983).) promoters. Suitable promoters for use in filamentous fungal host cells are, for example, the ADH3 / McKnight promoter, the EMBO J. 4, 2093 (1985), or the tpiA promoter.

The DNA sequence encoding the Kunitz-type TFPI variant I of the invention may also be operably linked to a suitable terminator, such as human growth hormone terminator (Palmiter et al., Supra) or (for fungal hosts) TPI1 (Alber and Kawasaki: supra) or ADH3 (McKnight et al., supra) promoters. This vector may further comprise elements such as polyadenylation signals (eg, from the SV 40 or the Elb region of adenovirus 5), transcriptional enhancement sequences (eg, the SV 40 enhancer), and translational enhancement sequences (eg, those encoding VA RNA adenovirus) ).

The recombinant expression vector of the invention may further comprise a DNA sequence that allows the vector to replicate in the appropriate host cell. Examples of such a sequence (if the host cell is a mammalian cell) are the SV 40 origin of replication or (if the host cell is a yeast cell) the yeast plasmid 2μ replication genes REP 1-3 and the origin of replication. The vector may also contain a selectable marker, eg, a gene whose product complements a defect in a host cell, such as a gene encoding dihydrofolate reductase (DHFR) or one that confers drug resistance, eg, neomycin, hygro * mycin or methotrexate, or Schi sesaccharomyces pombe TPI gene (described by PR Russell: Gene 40. 125 (1985)).

The methods used to ligate the DNA sequences encoding the Kunitz-type TFPI variant I of the invention, the promoter and the terminator, and methods of inserting them into suitable vectors containing vectors necessary for replication are well known to those skilled in the art (see, eg, Sambrook and Co .: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

The host cell into which the expression vector of the invention is introduced can be any cell that is capable of producing a Kunitz-type TFPI variant I of the invention. Preferably, the cell is a eukaryotic cell, such as a mammalian, yeast or fungal cell.

The yeast organism used as the host cell of the invention may be any yeast organism that produces large amounts of Kunitz-type TFPI domain I variant of the invention in culture. Examples of suitable yeast organisms are yeast strains of the species Saccharomvces cerevisiae, Saccharomyces kluyveri. Schi zosaccharomyces pombe or Saccharomvces uvarum. Transformation of yeast cells can be accomplished, for example, by protoplast formation and subsequent transformation, which is known per se.

Examples of suitable mammalian cell lines are COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10) or CHO (ATCC CCL 61) cell lines. Methods for transfecting mammalian cells and for expressing DNA sequences introduced into cells are described, for example, by Kaufman and Sharp: J. Mol. Biol. 159. 601 (1982) by Southern and Berg: J. Mol. Appl. Genet. 1, 327 (1982), Loyter et al., Proc. Nati. Acad. Sci. USA 79, 422 (1982), Wigler et al., Cell 14/725 (1978), Corsar and Pearson: Somatic Cell Genetics 2/603 Cl98lX _by Graham and van der Eb: Virology 52, 456 (1973) and Neuy Mann et al., EMBO J. 1,841 (1982).

Fungus cells can also be used as host cells of the invention. Examples of suitable fungal cells are filamentous fungal cells, e.g. Aspergillus sp. or Neurospora sp., in particular strains of Aspergillus oryzae or Aspergillus niger. Use of Aspergillus sp. for the expression of proteins is described, for example, in European patent 238 023.

The present invention further relates to a method of producing a Kunitz-type domain TFPI varinate according to the invention, a method of culturing cells as described above under conditions promoting expression of the variant and isolation of the resulting culture variant.

The medium used to cultivate the cells may be any conventional medium suitable for the growth of mammalian or fungal cells (including yeast cells), depending on the host cell being selected. The variant is secreted by the host cells into the growth medium. This medium is isolated by conventional techniques, such as separating cells from the medium by centrifugation or filtration, precipitating the protein components of super11 natant or salt filtrate, e.g., ammonium sulfate, purification by various chromatography techniques, e.g., ion-exchange or affinity chromatography, or similar procedures.

The present invention also relates to a pharmaceutical composition comprising a Kunitz-type TFPI variant I of the invention together with a pharmaceutically acceptable carrier or excipient. In preparing the composition of the invention, the variant is processed by any known means of preparing the pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences, 1985. The composition is typically prepared in a form suitable for systemic injection or infusion, and may be prepared as a sterile solution. water, isotonic saline or glucose solution.

Surprisingly, it has been found that TFPI Kunitz-type * domain I by itself is capable of inhibiting cathepsin G. The present invention therefore also relates to a pharmaceutical composition for inhibiting cathepsin G, a composition comprising human Kunitz-type TFPI protease inhibitor domain I or variant thereof as above. described and a pharmaceutically acceptable carrier or excipient.

The Kunitz-type TFPI variant I of the invention is therefore preferably intended for use in those therapeutic applications that envisage the use of natural aprotinin or aprotinin analogues with other inhibitory profiles, especially when large doses of aprotinin are required. Therapeutic applications for which the use of the variants of the invention are indicated as a result of their ability to inhibit human serine proteases such as trypsin, plasmin, kallikrein, elastase, cathepsin G and proteinase-3 include, but are not limited to, acute pancreatitis, inflammation , thrombocytopenia, platelet function protection, organ protection, wound healing, shock (including pulmonary shock) and conditions including hyperfibrinolytic haemorrhage, emphysema, rheumatoid arthritis, adult respiratory distress syndrome, chronic inflammatory bowel disease and psoriasis, in other words, o which are believed to be caused by pathological proteolysis elastase, cathepsin G and proteinase-3 released from activated PMNs.

Furthermore, the present invention relates to the use of a TFPI domain I inhibitor of the Kunitz type or a variant thereof as described above for the preparation of a medicament for the prevention or treatment of diseases or conditions associated with pathological proteolysis proteases released from overstimulated PMNs. As mentioned above, it may be advantageous to administer heparin concurrently with the TFPI domain I of a Kunitz-type inhibitor or a variant thereof.

In addition to the aforementioned pharmaceutical use, Kunitz-type TFPI domain II or a variant thereof as described above can be used to isolate natural substances, e.g., proteases or receptors, from human material that bind directly or not directly to T-FPI. domain-II-Kunitzova. type, "eg. testing or affinity chromatography.

The invention is further illustrated by the following examples, which are in no way intended to limit the scope of the invention as claimed in the claims.

DETAILED DESCRIPTION OF THE INVENTION

General methodology

Standard DNA techniques were performed as described in the literature (SambrQok J .- Fritsch E .F ...... a.

Molecular Cloning: A Laboratory Manual ^'1, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Synthetic oligonucleotides were prepared on an automated DNA synthesizer (380B, Applied Biosystems) by phosphoramidite chemistry on a controlled pore size glass support (Beaucage SL and Caruthers ΜH: Tetrahedron Letters 22, 1859 (1981)). DNA sequences were determined by the dideoxy technique (Sanger F., Micklen S., Coulson AR: Proc. Natl. Acad. Sci. USA 74, 5463 (1977).). Polymerase chain reactions (PCR) were performed on a DNA Thermal Cycler (Perkin Elmer Cetus),

Amino acid analysis was performed after hydrolysis in 6M HCl at 110 ° C in a tube (sealed under vacuum) for 24 hours. The analyzes were performed on a Beckmam 121MB automatic amino acid analyzer modified for micro-scale work.

N-terminal amino acid sequencing analysis was performed by automated Edman degradation with an Applied Biosystems 470A gas-phase sequencer. Detection and quantitation of released pTH amino acids from each sequencing cycle is performed by continuous reverse phase HPLC analysis.

Molecular weights were determined on a plasma desorption (PDMS) BIO-ION 20 mass spectrometer with an airborne tube of approximately 15 cm in length operating in a positive mode. The 5μ1 fractions were analyzed at an accelerating voltage set at 15 kV. Ions corresponding to 5 million fission were isolated. For well-defined maxima, the accuracy of molecular ion assignment is approximately 0.1%, in other cases it is somewhat lessΓ

Example 1

Production of the First Kunitz Domain Tissue Factor Inhibitor, TFPI-1 from Yeast KFN-1651 cDNA cDNA encoding full-length TFPI is isolated from human liver-derived HepG2 (ATCC HB 8065) and inserted as a BamHI-XbaI 900 bp fragment into the mammalian expression vector pKFN-1168 as described in the literature (Pedersen AH, Nordfang O., Norris F., Wiberg FC, Christensen, Ρ., Moeller, Β., Meidahl-Pedersen, J., Beck TC, Norris. K., Hedner U., Kisiel W., J. Biol. Chem., 265, 16786 (1990). The DNA sequence of this insert is shown in SEQ ID NO. TFPI-1 is encoded by nucleotides 152 to 325 as indicated.

TFPI-1: 0.1 yq BamHI-XbaI 900 bp fragment from pKFN-1168 was used as a template in a PCR reaction with 100 pmoles of both primers: Nor-2524 (GCTGAGAGATTGGAGAAGAGAATGCATTCATTTTGTGC) and NOR-2525 (TAATCCTTCTGATTA). The 17 nucleotides at the 3 'end of NOR-2524 are identical to nucleotides 152 to 168 in the TFPI-1 gene of SEQ ID NO: 1. Nos. 3 and 21 of the nucleotides at the 5'-end are identical to nucleotides 215 to 235 in the synthetic leader gene (vi2 of SEQ ID NO: 5) from the above described pKFN-1000. Primer NOR-2525 is complementary to nucleotides 311 to 325 of SEQ ID NO. No. 3 and has a 5 'extension containing a translational stop codon followed by an XbaI site.

The PCR reaction was performed in a volume of 100 μΐ using a commercial kit (GeneAmp, Perkin Elmer Cetus) in the following cycle: 94 ° C for 20 seconds, 50 ° C after. for 20 seconds and 72 ° C for 30 seconds. After 19 cycles, a final cycle was performed. wherein the 72 ° C stage was maintained for 10 minutes. The PCR product, a fragment of 211 bp, was isolated by electrophoresis on

2% agarose gel.

Signal-leader: 0.1 μς of the PvuII fragment of the 700 bp nucleotide from the pKFN-1000 described below is used as a template in a PCR reaction containing 100 pmoles of both primers: NOR-1478 (GTAAATCGACGGCCAGT) and NOR-2523 (TCTCTTCTCCAATCTCTCAGC). NOR-1478 matches the upstream sequence from the EcoRI site of SEQ ID NO: 1. Primer NOR-2523 is complementary to 17 nucleotides at the 3 'end of the synthetic leader gene of plasmid pKFN-1000, see SEQ ID NO. The PCR reaction was performed as above. This yields a —257- nucleotide pair fragment ......................... — _____ ____

Plasmid pKFN-1000 is a derivative of plasmid pTZ19R (Mead D.A., Szczesna-Skorupa E. and Kemper B .: Prot. Engin. I, 67 (1986)) containing DNA encoding a synthetic yeast signal leader peptide.

Plasmid pKFN-1000 is described in WO 90/10075. A DNA sequence of 235 bp downstream of the EcoRI site of plasmids pKFN-1000 and the encoded amino acid sequence of the synthetic yeast leader is set forth in SEQ ID NO. No 5.

Signal-leader-TFPI-1: Mix approximately 0.1 μς of the two PCR fragments described above. A PCR reaction was performed with 100 pmoles of both NOR-1478 and NOR-2525 primers and the following cycle: 94 ° C for 1 minute, 50 ° C for two minutes and 72 ° C for three minutes. After 16 cycles, a final cycle is performed in which the temperature of 72 ° C is maintained for 10 minutes.

The resulting 443 bp fragment was purified by 1% agarose gel electrophoresis. It was then digested with EcoRI and XbaI. The resulting 412 bp fragment was ligated with the 9500 bp NcoI-XbaI fragment of pMT636 and the 1400 bp NcoI-EcoRI fragment of pMT636. Plasmid pMT636 is described in WO 89/01968. '*. n. ·

PMT636 is an E. coli - S. cerevisiae shuttle vector for multiple hosts containing the Schizosaccharomyces pombe TPI gene (POT) (Russell, PR, Gene 40 125 (1985).) S. cerevisiae triosefos- ^w fat-isomerase promoter and terminator, TPI , and TPI _T (Alber T. á · · ''ů *

Kawasaki, G .: J. Mol. Appl. Gene. 1, 419 (1982).

This ligation mixture was used to transform the appropriate E. coli strain (r, m ⁺ ) selected for ampicillin resistance.

DNA sequencing showed that plasmids from the resulting colonies contained the correct DNA sequence for TFPI-1 correctly fused to the synthetic yeast signal-leader gene.

One plasmid pKFN-1603 is selected for further use. The construction of plasmids pKFN-1603 is illustrated in Figure 1.

The plasmid expression cassette pKFN-1603 contains the following sequence:

TPI _P - KFN1000 signal-leader - TFPI1 - TPI _T.

The DNA sequence of the 412 bp EcoRI-XbaI fragment of pKFN-1603 is set forth in SEQ ID NO. C. 7.

Yeast Transformation: S. cervisiae strain MT663 (E2-7B XE11-36, α / α, deltatpi / deltatpi, pep 4-3 / pep 4-3) was grown to YPGaL (1% Bacto yeast extract, 2% Bacto peptone) , 2% galactose, 1% lactate) to an optical density of 0.6 at 600 nm.

100 ml of the culture was treated by centrifugation, washing with water (10 ml), centrifuging again and resuspending in 10 ml of a solution containing 1.2 M sorbitol, 25 mM Na _with EDTA, pH 8.0, and 6.7 mg / ml dithiothreitol. This suspension is incubated for 15 minutes at 30 ° C, centrifuged and the cells are resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na ₂ EDTA, 0.1 M sodium citrate, pH 5.8, and 2 mg Novozyme ^® R ¹³⁴ . the suspension is incubated for 30 minutes. . at 30 ° C, both the cells and the sol are eaten. by centrifugation, _L. with 10 ml of 1.2M sorbitol and 10 ml of CAS (1.2M sorbitol, 10mM calcium chloride, 10mM Tris HCl (Tris means tris (hydroxymethyl) aminomethane, pH 7.5) and resuspended in 2ml CAS. resuspended in CAS are mixed with approximately 0.1 µg of plasmids pKFN-1603 and left at room temperature for 15 minutes, 1 ml of the mixture (20% polyethylene glycol 4000, 20 mM calcium chloride, 10 mM calcium chloride, 10 mM Tris HCl, pH 7.5) is added. The pellet is resuspended in 0.1 ml SOS (1.2 M sorbitol, 33% (v / v) YPD, 6.7mM chloride) and the mixture is allowed to stand for 30 minutes at room temperature. calcium (14 mg / ml leucine) and incubated for 2 hours at 30 DEG C. The suspension is centrifuged and the pallet is resuspended in 0.5 ml of 1.2 M sorbitol.

-----at-5 ° C - at 6 ° C overhead agar (SC medium, see Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory ( 1982) containing 1.2 M sorbitol and 2.5% agar) and the suspension is poured onto the surface of plates containing solidified agar and sorbitol containing medium.

Transformed colonies were harvested after 3 days at 30 ° C, collected again and used to start liquid cultures. One of these transformants KFN-1651 is selected for characterization.

Fermentation: Yeast strain KFN-1651 was grown on YPD medium (1% yeast extract, 2% peptone (from Difco Laboratories) and 3% glucose). One liter of culture of this strain is shaken at 30 ° C to an optical density of 24 at. 650 nm. After centrifugation, the supernatant was collected.

The pH of the yeast supernatant was adjusted to 3.0 with 5% acetic acid and phosphoric acid, applied to a S-Sepharose Fast Flow (Pharmacia) column and equilibrated with 50 mM formic acid, pH 3.7. After washing with equilibration buffer, the HKI-domain is eluted with 1 M sodium chloride. Desalting was performed on a Sephadex G-25 (Pharmacia) column equilibrated and eluted with 0.1% ammonium bicarbonate, pH 7.9. After concentration by centrifugation under vacuum and pH adjustment to 3.0, further

T * purification is performed on a Mono S column (Pharmacia) equilibrium; bridged with 50 mM formic acid, pH 3.7. After washing with equilibration buffer, a gradient elution from 0 to 1M sodium chloride in equilibration buffer is used. Final purification was performed on a Vydac C4 column (The Separation Group, Ka., USA) by reverse phase HPLC eluting from 5 to 55% acetonitrile to 0.1% TFA. The purified product is lyophilized by centrifugation in vacuo and redissolved in water.

Aliquots were analyzed by mass spectrometry (molecular weight: found: 6853.5, calculated: 6853.8) and amino acid sequencing at the N-terminus of 45 Edman degradation cycles, which confirmed the primary structure of the TFPI-1 domain (Table i) .

Table 1

N-terminal sequencing analysis of TFPI-1

Approximately 350 pmoles of KFN1651 (HPLC fraction 18/920327) are analyzed.

The repetitive yield is xx, x%.

cycle C. aminoky- selina yield (pmoles) cycle C. aminoky- selina yield (pmoles) 1 Met 250 31 Glu 19 Dec 2 His 47 32 Glu 25 _____ 3 ™ ____Ser______ 69 33. __Phe_ 27 Mar: 4 Phe 301 34 Ile 18 5 Cys - 35 Tyr 16 6, Ala 226 36 Gly 26 7 Phe 201 37 Gly 36 8 Lys 201 38 Cys - 9 Ala 216 39 Glu 11 10 Asp 105 40 Gly 25 11 Asp 117 41. Asn 14 12 Gly 148 42 Gin 15 Dec 13 For 62 43 Asn 19 Dec 14 Cys 44 Arg 15 Dec 15 Dec Lys 78 45 Phe 12

-16 -Ala— 9 8 ~ —_— -____ 46 .................. Gl.U

17 Ile 75 47 Ser 18 Met 57 48 Leu 19 Dec Lys ⁶⁹ 49 Glu 20 May Arg 48 50 Glu 21 Phe 63 51 Cys 22nd Phe 90 52 Lys 23 Phe 99 53 Lys 24 Asn 46 54 Met

Table 1 (continued)

cycle aminoky- yield cycle aminoky- yield C. selina (pmoles) C. selina (pmoles) 25 Ile 50 55 Cys 26 Phe 56 56 Thr 27 Mar: Thr 25 57 Arg 28 Arg 35 58 Asp 29 Gin 33 30 Cys -

The CTH PTH derivative is not identified, eg, cycles 5, 14, 30 and 38.

The sequencer was stopped after 60 cycles. The sequence could be derived for the first 45 amino acids.

Example 2

Inhibition of TFPI serine proteinases (domain I) of KFN 1651 yeast culture medium is purified by KFN 1651. The concentration of KFN 1651 is determined from absorbance at 214 nm with BPTI as standard. Porcine trypsin and human recombinant factor VIIa were obtained from Novo Nordisk A / S (Bagsvaerd, Denmark), bovine chymotrypsin (TLCK-treated) was obtained from Sigma Chemical Co. (St. Louis, Mo., USA). Human truncated recombinant tissue factor was obtained from Corvas (San Diego, CA).

Human neutrophil cathepsin G is purified from PMN extracts according to the method described by Baugh and Travis (Biochemistry 15, 836 (1876)). Peptidyl nitroanilide substrates S2251, S2586 and Ξ2288 are obtained from Kabi (Stokholm, Sweden), S7388 is obtained from Sigma Chemical Co. (St. Louis, Mo., USA) and FXa-1 were obtained from NycoMed (Oslo, Norway).

Serine proteinases are incubated with various concentrations of KFN 1651 for 30 minutes. Substrate is then added and the resulting proteinase activity is measured at 405 nm. The results are shown in Table 2.

The unmodified TFPI Kunitz domain I (KFN 1651) was found to be an inhibitor of trypsin, chymotrypsin, neutrophil cathepsin G, and factor VIIIa / tissue factor.

Table 2

proteasa Apparent Ki trypsin 18.10 ’M ------- chymotrypsin _______. _________________________ cathepsin G 87.10 ’M Factor VIIa / TF 150.10 ’M

List of sequences (1) General information:

(i) Applicant:

(A) Name: Novo Nordisk A / S (B) Street: Novo Alle (C) City: Begsvaerd (E) Country: Denmark (F) Zip / Postal Code: DK-2880 (G) Telephone: +45 4444 8888 (H ) fax: +45 4449 3256 (I) telex: 37304 (ii) Title of the invention: Human protease inhibitor variant

Kunitz type (iii) Number of sequences: 8 (iv) Computer read form:

(A) media type: floppy disk (B) computer: IBM PC compatible (C) operating system: PC-DOS / MS-DOS (D) computer program (software): Patentln Release

No. 1.0, version No. 1.25 (EPO) (2) Sequence Information id. no. 1:

(i) Sequence characteristics:

(A) length: 55 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (vi) original source:

(A) organism: synthetic (xi) sequence description: sequence id. no. 1:

Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 15 10 15

Xaa Xaa Phe Phe Phe Asn Ile Phe Thr

25 30

Xaa Tyr Gly Gly Cys Xaa Xaa Xaa Gin Asn Arg Phe Xaa Ser Leu

40 45

Glu Glu Cys Xaa Xaa Met Cys Thr Arg Xaa

55 (2) Sequence Information id. No. 2:

(i) Sequence characteristics:

(A) length: 58 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (vi) original source:

(A) organism: synthetic (xi) sequence description: sequence id. C. 2:

MetHis -Ser Phe CysAlaPhe Lys Ala Asp Asp Gly Tro .Cys.-LYS 1 5 1015

Ala Arg Ile Ile Arg Phe Phe Phe Asn Ile Phe Thr Arg Gin Cys 20 25 30

Glu Glu Phly Val Tyr Gly Gly Cly Arg Ala Lys Gin Asn Arg Phe 35 40 45

Glu Ser Leu Glu Clu Lys Lys Met Cys Thr Arg Asp 50 55 (2) Sequence Id. No. 3:

(i) Sequence characteristics:

(A) length: 945 nucleotide pairs (B) type: nucleic acid (C) fiber type: single-stranded (D) topology: linear (ii) molecule type: cDNA (vi) original source:

(A) organism: Homo sapiens (ix) properties:

(A) Name / Key: CDS.

(B) location: 152..325 (xi) sequence description: sequence id. No. 3:

GGATCCGAAT TCCACCATGA AGAAAGTACA TGCACTTTGG GCTTCTGTAT 50

GCCTGCTGCT TAATCTTGCC CCTGCCCCTC TTAATGCTGA TTCTGAGGAA 100 'á

GATGAAGAAC ACACAATTAT CACAGATACG GAGTTGCCAC CACTGAAACT 150

T ATG CAT TCA TTT TCA GTC TAG AAG GCG GAT GAT GGC CCA TGT 193 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys

10

ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATT ATA ATT ATA ATT ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATA ATT ATA TTC ACT CGA 235

Lys Ala Ile Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg

20 25

CAG TGC GAA GAA TTT ΑΤΑ TAT GGG GGA

Gin Gys Glu Glu Gly Gly Gly Gly Gly Gly

35 40

AAT CGA TTT GAA AGT ATG TGT ACA 319

Asn Arg Phe Glu Ser Clu Lys Met Cys Thr

50 55

AGA GAT AATGCAAACA GGATTATAAA GACAACATTG CAACAAGAAA 365

Arg Asp

AGCCAGATTT CTGCTTTTTG GAAGAAGATC CTGGAATATG TCGAGGTTAT 415

ATTACCAGGT ATTTTTATAA CAATCAGACA AAACAGTGTG 465 _________GTATGGTGGA -TGCCTGGGCA - ATATGAACAA JTTTTGAGACA CTGGAAGAAT. _ 515

CGAAGAACAT TTGTGAAGAT GGTCCGAATG GTTTCCAGGT GGATAATTAT 565

GGAACCCAGC TCAATGCTGT GAATAACTCC CTGACTCCGC AATCAACCAA 615

GGTTCCCAGC CTTTTTGAAT TTCACGGTCC CTCATGGTGT CTCACTCCAG 665

CAGACAGAGG ATTGTGTCGT GCCAATGAGA ACAGATTCTA CTACAATTCA 715

GTCATTGGGA AATGCCGCCC ATTTAAGTAC AGTGGATGTG GGGGAAATGA 765

AAACAATTTT ACTTCCAAAC AAGAATGTCT GAGGGCATGT AAAAAAGGTT 815

TCATCCAAAG AATATCAAAA GGAGGCCTAA TTAAAACCAA AAGAAAAAGA 865

AAGAAGCAGA GAGTGAAAAT AGCATATGAA GAGATCTTTG TTAAAAATAT 915

GTGAATTTGT TATAGCAATG TAACTCTAGA 945 (2) Sequence Information id. 4:

(i) Sequence characteristics:

(A) length: 58 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (xi) sequence description: sequence id. 4:

Met His Ser Phys Cys Ala Phys Lys Ala Asp Asp Gly Pro Cys Lys 15 10 15

Ala Ile Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gin Cys

25 30

Glu Gly Gly Gly Gly Gly Gly Gly

40 45

Glu Ser Leu Glu Cys Lys Met Lys Met Cys Thr Arg Asp

55 (2) Sequence Information id. No 5:

(i) Sequence characteristics:

(A) length: 235 pairs of nucleotides (B) type: nucleic acid (C) fiber type: single-stranded (D) topology: linear (ii) molecule type: cDNA (vi) original source:

(A) organism: synthetic (ix) properties:

(A) name / key: CDS (B) location: 77..235 (xi) sequence description: sequence id. No. 5:

GAATTCCCATT CAAGAATAGT TCAAACAAGA AGATTACAAA CTATCAATTT 50

Catacacat ATAAACGACC AAAAGA ATG AAG GCT GTT TTC TTG GTT 97'Met Lys Ala Val Phe Leu Val

5

TTG TCC TTG ATC GGA TTC TGC TGG GCC CAA CCA GTC ACT GGC 139 Leu Ser Leu Ile Gly Phe Cys Trp Ala Gin Pro Rol Thr Gly - 10 15 Dec 20 May -GAA- -TCA -TCT -GTT- GAG.ATTCCG GAA GAG TCT CTG ATC ATC 181 Asp Glu Ser Ser Wall Glu Ile Pro Glu Glu Ser Leu Ile Ile 25 30 35 GCT GAA AAC ACC ACT TTG GCT AAC GTC GCC ATG GCT GAG AGA 223 Ala. GlU Asn Thr Thr Leu Ala Asn Wall Ala Met Glu Arg 40 45 TTG GAG AAG AGA AND 235 Leu Glu Lys Arg 50 (2) Information about SEQ ID NO. C. 6:

(i) Sequence characteristics:

(A) length: 53 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein

(xi) sequence description: sequence id. No. 6:

Met Lys Ala Wall Phe Leu Wall Leu Ser Leu Ile Gly Phe Cys Trp E 1 5 10 15 Dec - Ala Gin For Wall Thr Gly Asp Glu Ser Ser Wall Glu Ile For Glu 20 May 25 30 Glu Ser Leu Ile Ile Ala Glu Asn Thr Thr Leu Ala Asn Wall Ala 35 40 45 Met Ala Glu Arg Leu Glu Lys Arg 50

(2) Sequence information id. No 7:

(i) Sequence characteristics:

(A) length: 418 nucleotide pairs (B) type: nucleic acid (C) fiber type: single-stranded (D) topology: linear (ii) type of molecule; cDNA (vi) original source:

(A) organism: synthetic / human /

(ix) Features:

(A) Name / Key: CDS (B) Location: 77..409 (ix) Features:

(A) Name / Key: sig_peptid (B) Location: 77..235 (ix) Features:

(A) name / key: mat_peptid (B) location: 236..409 (xi) sequence description: sequence id. No. 7:

GAATTCCATT CAAGAATAGT TCAAACAAGA AGATTACAAA CTATCAATTT 50

CATACACAAT ATAAACGACC AAAAGA ATG AAG GCT GTT TTC TTG GTT 97

-53 -50

TTG TCC TTG ATC Leu lle CGA TTC TGC GCC GCC CAA CCA GTC ACT GGC 139 Leu Ser -45 Gly Phe Cys -40 Trp Ala Gin Pro Val -35 Thr Gly GAT GAA -Glu- TCA Ser TCT Ser GTT -Wall GAG -Glu- ATT lle- CCG -For- GAA Glu- GAG -Glu- TCT -Ser- CTG Leu ATC -He- ATC lle— 181 ~ Asp -30 -25 -20 GCT GAA AAC ACC ACT TTG GCT AAC GTC GCC ATG GCT GAG AGA 223 Ala Glu Asn Thr Thr Leu Ala Asn Wall Ala Met Ala Glu Arg -15 -10 -5 TTG GAG AAG AGA ATG CAT TCA TTT TGT GCA TTC AAG GCG GAT 5 Leu Glu Lys Arg Met His Ser Phe Cys Ala Phe Lys Ala Asp 1 5 10 GAT GGC CCA TGT AAA GCA ATC ATG AAA AGA TTT TTC TTC AAT 307 Asp Gly For Cys Lys Ala lle Met Lys Arg Phe Phe Phe Asn

15 ...............-....... ^: ............... ......................'...... 2 0

ATT TTC ACT CGA CAG TGC lle Phe Thr Arg. Gin Cys GAA Glu GAA TTT ΑΤΑ TAT GGG GGA TGT Glu Phe lle tyr gly 35 Gly Cys 25 30 GAA GGA AAT CAG AAT CGA TTT GAA AGT CTG GAA GAG TGC AAA Glu Gly Asn Gin Ash Arg Phe Glu Ser Leu Glu Glu Cys Lys 40 45 50

. tk

AAA ATG TGT AGA GAT TAATCTAGA 418

Lys Met Cys Thr Arg Asp (2) Sequence Information id. No. 8:

(i) Sequence characteristics:

(A) length: III amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (xi) sequence description: sequence id. No. 8:

Met -53 Lys Ala Phe Leu Val Leu Ser Leu Ile Gly Phe Cys -35 Trp -50 -45 Ala Gin For wall Thr Gly Asp Glu Ser Ser Wall Glu Ile For Glu -30 -30 -25 Glu Ser Leu Ile Ile Ala Glu Asn Thr Thr Leu Ala Asn Wall Ala -20 -15 -10 Met Ala Glu Arg Leu Glu Lys Arg Met His Ser Phe Cys Ala Phe -5 1 5 Lys Ala Asp Asp Gly For Cys Lys Ala Ile Met Lys Arg Phe Phe 10 15 Dec 20 May Phe Asn Ile Phe Thr Arg Gin Cys Glu Glu Phe Ile Tyr Gly Gly 25 30 35 Cys Glu Gly Asn Gin Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys 40 45 50

Lys Met Cys Thr Arg Asp

Claims (38)

PATENT CLAIMS 1. A Kunitz-type tissue factor inhibitor (TFPI) protease inhibitor human domain I variant comprising the following amino acid sequence X ¹ Cys Ala Phe Lys Ala Asp X ² Gly X ³ Cys X * X ⁵ X ⁶ X ⁷ X ⁸ X ⁹ Phe Phe Phe Asn lle Phe Thr Arg Gin Cys Glu Glu Phe X ¹⁰ Tyr Gly Gly Cys X ¹¹ X ¹² X ¹³ Gln Asn Arg Phe X "Ser Leu Glu Glu Cys X ^{X 5} X ^ls Met Cys Thr Arg X ^17, wherein X ¹ is hydrogen or a 1-7 residue naturally occurring amino acid except Cys, X ² to X ¹⁶ are independently a naturally occurring amino acid residue thereon, and X ¹⁷ is a hydroxyl group or 1-5 naturally occurring amino acid residues except Cys, with at least one of X ¹ to X ¹⁷ amino acid residues other than the corresponding amino acid residue of the natural sequence . A variant according to claim 1, characterized in that X ¹ is Ser-Phe or Met-His-Ser-Phe. 3. A variant according to claim 1, characterized in that X ² is an amino acid residue which is selected from a group consisting of Ala, Arg, Thr, Asp, Pro, Glu, Lys, Gln, Ser, lle and Val. 4. A variant according to claim 3, characterized in that X ² is Thr or Asp. 5. A variant according to claim 1, characterized in that X ³ represents an amino acid residue selected from the group consisting of Pro, Thr, Leu, Arg, and Val. Ile. 6. A variant according to claim 5, characterized in that X ³ is Pro or Ile. 7. A variant according to claim 1, wherein X * is an amino acid residue selected from the group consisting of Lys, Arg, Val, Thr, Ile, Leu, Phe. Gly, Ser, Met, Trp, Tyr, Gln, Asn, and Ala. A variant according to claim 7, wherein X ⁴ is Lys, Val, Leu, Ile, Thr, Met, Gln or Arg. A variant according to claim 1, wherein X ⁵ is an amino acid residue selected from the group consisting of Ala, Gly, Thr, Arg, Phe, Gln and Asp. 10. A variant according to claim 9, wherein X is ^s. means Ala, Thr, Asp or Gly. 11. A variant according to claim 1, characterized in that X ⁶ is an amino acid residue selected from the group consisting of Arg, Ala, Lys, Leu, Gly, His, Ser, Asp, Gln, Glu, Val, Thr, Tyr, Phe, Asn, Ile, and Met. A variant according to claim 11, wherein X ⁶ is Arg, Phe, Ala, Ile, Leu or Tyr. A variant according to claim 1, wherein X ⁷ is an amino acid residue selected from the group consisting of: Met-GlnT-GlU7 "Thr7" LeuT-Val ---------- -a Phe. and The variant of claim 13, wherein X ⁷ is Ile. 15. A variant according to claim 1, wherein X is an amino acid residue selected from the group consisting of Ile, Thr, Leu, Asp, Lys, Ser, Gln, Glu, Arg, Pro and Phe, A variant according to claim 15, wherein X ⁸ is Ile or Lys. 17. The variant of claim 1 wherein X 'is an amino acid residue selected from the group consisting of Arg, Ser, Ala, Gln, Lys, and Leu. The variant of claim 17, wherein X 'is Arg. 19. A variant according to claim 1, characterized in that X ^J0 represents an amino acid residue selected from the group consisting of Gln, Pro, Phe, Ile, Lys, Trp, Ala, Thr, Leu, Ser, Tyr, His, Asp, Met, Arg and Val. ' 20. A variant according to claim 19, wherein X ¹⁰ is Val or Ile. 21. A variant according to claim 1, characterized in that X ¹¹ represents ^an amino acid residue selected from the group consisting of Gly, Met, Gln, Glu, Leu, Arg, Lys, Pro and Asn. 22. A variant according to claim 21, wherein X ¹¹ is Arg or Glu. The variant of claim 1, wherein X ¹² is Ala or Gly. 24. The variant of claim 1, wherein ^X13 is an amino acid residue selected from the group consisting of Lys, Asn, and Asp. 25. A variant according to claim 24, wherein X ¹³ is Lys or Asn. 26. A variant according to claim 1, wherein ^X14 is an amino acid residue selected from the group consisting of 2 Val, Tyr, Asp, Glu, Thr, Gly, Leu, Ser, Ile, Gin, His, Asn, Pro, Phe, Met, Ala, Arg, Trp, and Lys. 27. A variant according to claim 26, wherein X ¹⁴ is Lys or Glu. 28. Variant according to claim 1, characterized by j í c Leu. and s e by X ¹⁵ is Lys, Met, Glu or 29. Variant according to Claim 1 on č u j í c and s e by that X ^lfi means Lys, Ala, Asn or Glu. -Varřanta-30 ™ according to claim Ty ~ ------ ~ T ~~ characterized 'in that X ¹⁷ is Asp. The variant of claim 1, wherein X ¹ is Met-His-Ser-Phe and X ¹⁷ is Asp. 32. The variant of claim 1 comprising the following amino acid sequence: Met His Ser Phy Cys Ala Phy Lys Ala Asp Asp Gly Pro Cys Lys Ala Arg Ile Ile Arg Phe Phe Phe Asr Ile Phe Thr Arg Gin Cys Glu Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr _-ArgAspCsesekid‘‘‘‘‘‘) 2). 33. A DNA construct comprising a DNA sequence encoding a human variant of a Kunitz-type protease inhibitor according to any one of claims 1 to 32. 34. A recombinant expression vector comprising the DNA construct of claim 33. 35. A cell comprising the DNA construct of claim 33 or the expression vector of claim 34. A method of producing a human variant of a Kunitz-type protease inhibitor according to any one of claims 1 to 32, comprising culturing the cell of claim 35 under conditions conducive to protein expression and isolating the resulting protein from the culture. A pharmaceutical composition comprising a human variant of a Kunitz-type protease inhibitor according to any one of claims 1 to 32 and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition for the inhibition of cathepsin G, comprising human domain I Kunitz-type protease inhibitor TFPI or a variant thereof according to any one of claims 1 to 32 and a pharmaceutically acceptable carrier or excipient. The composition of claim 37 or 38, further comprising heparin. Use of human domain I Kunitz-type protease inhibitor TFPI or a variant thereof according to any one of claims 1 to 32 for the preparation of a medicament for the prevention or treatment of diseases or conditions associated with pathological proteolysis.