DE102007056231A1 - Proteins comprising a Kunitz domain useful as protease inhibitors for therapeutic purposes - Google Patents

Abstract

Proteins comprising a Kunitz domain with the following amino acids at positions numbered according to aprotinin are new: X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = H, X19 = P, X20 = R, X21 = W, X22 = W; or X10 = V, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F; or X10 = E, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F; or X10 = V, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F; or X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F. Independent claims are also included for: (1) fragment of a protein as above with an inhibitory activity against plasmin, plasma kallikrein or factor XIa at least comparable to that of aprotinin; nucleic acid coding for a protein as above; (2) cell containing the nucleic acid; (3) nonhuman organism containing the nucleic acid; (4) producing a protein as above by culturing a cell as above. ACTIVITY : Hemostatic; Anticoagulant; Vasotropic; Antibacterial; Immunosuppressive; Antianginal; Cardiant; Cerebroprotective; Antiinflammatory; Antirheumatic; Antiasthmatic; Cytostatic; Analgesic; Vulnerary. MECHANISM OF ACTION : Protease inhibitor. A protein comprising SEQ ID NO:121 (defined sequence of 58 amino acids given in the specification) had IC50 values (nM) of 2 against trypsin, 229 against plasmin, 1919 against factor XIa and 287 against plasma kallikrein.

Description

The The present invention relates to protease inhibitors of the Kunitz type, isolated as DNA fragments from human genomic DNA whose modification and expression in suitable expression systems, as well as their production and possible use.

Kunitz domains are polypeptides of 55-62 amino acids (aa) in length that inhibit a variety of different potency serine proteases. They usually contain three disulfide bridges, which stabilize the protein and determine its three-dimensional structure. The interaction with the respective serine protease occurs mainly via an approximately 9 aa long loop in the N-terminal region of the Kunitz domain. This loop binds to the catalytic center of the protease and thus prevents the cleavage of the corresponding protease substrates ( Laskowski and Kato, 1980; Bode and Huber, 1992 ).

aprotinin, also called bovine pancreatic trypsin inhibitor (BPTI) becomes a prototype of the Kunitz domains (Fritz and Wunderer, 1983). It is a basic protein of 58 aa Length isolated from various organs of bovine (including the pancreas, lungs, liver and heart). Aprotinin will stabilized by three disulfide bridges (Cys 5-Cys 55; Cys 14-Cys 38; Cys 30-Cys 51) and is u. a. a potent inhibitor of trypsin, plasmin and plasma kallikrein.

X-ray crystallographic analysis of the aprotinin-bovine trypsin complex has shown that the aprotinin contact region to the protease catalytic center is essentially formed by a loop consisting of amino acid residues 11 through 19 (see Bode and Huber, 1992 and references therein). Of central importance for the inhibitory action of aprotinin is the amino acid residue Lys / K 15, which is in particularly close contact with the catalytically active serine residue of the protease. The amino acid Lys / K 15 is therefore designated by definition as P1 radical ( Schechter and Berger, 1967 ). N-terminal of Lys / K 15 are the residues P2, P3, etc., while the amino acids C-terminal of Lys / K15 are referred to as P1 ', P2', etc. It has previously been shown that the inhibitory effect of aprotinin can be altered by targeted replacement of amino acid residues in the range from residue 11 to residue 19 ( Otlewski et al., 2001 . Apeler et al., 2004 . Krowarsch et al., 2005 ). In addition, the amino acids at position 36-39 are important for the effectiveness of aprotinin ( Fritz and Wunder, 1983 . Krowarsch et al., 2005 ).

Aprotinin is now widely used in cardiac surgery under the trade name Trasylol, after clinical studies have shown that treatment with aprotinin significantly reduces the need for transfusion in such operations and leads to the reduction of rebleeding ( Royston, 1992 ). Its clinical effect is attributed to the inhibition of fibrinolysis, the inhibition of intrinsic blood coagulation (contact activation) and the reduction of thrombin formation ( Blauhut et al., 1991 . Dietrich et al., 1995 ). Thus, inhibition of both the plasma and plasma kallikrein and factor XIa is important for the hemostatic effect of aprotinin.

As a bovine protein, aprotinin in humans leads to the formation of antibodies. Repeated Trasylol may cause allergic reactions (anaphylactic shock). The risk is 2.8%, which limits the possibility of multiple use of aprotinin ( Dietrich et al., 2001 . Beierlein et al., 2005 ). There is thus a high medical need for active ingredients with a similar or better clinical effect than aprotinin, which do not cause an allergic reaction.

In the human genome exist a number of genes that contain one or more Kunitz domains (KD), such as. B. amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease, Ponte et al., 1988 . Oltersdorf et al., 1989 ), Serine peptidase inhibitor-like, with Kunitz and WAP domains 1 (Eppin, Richardson et al., 2001 ), Serine peptidase inhibitor, Kunitz type, 2, SPINT 2 (placental bikunin, 2 KD, Delaria et al., 1997 ), Tissue factor pathway inhibitor 2 precursor (TFPI-2, 3 KD, Sprecher et al., 1994 ), Tissue factor pathway inhibitor, TFPI (lipoprotein-associated coagulation inhibitor, 3 KD, Wun et al., 1988 . van der Logt et al., 1991 ), Amyloid beta (A4) precursor-like protein 2, APLP2 ( Yang et al., 1996 ), Alpha-1-microglobulin / bikunin precursor, AMBP, 2KD ( Vetr and Gebhard, 1990 ), Hepatocyte growth factor activator inhibitor typed, (HAI-1, SPINT 1, 2 KD, Shimomura et al., 1997 ), Collagen, type VI, apha 3 (COL6A3, Chu et al., 1988 ), Collagen, type VII, apha 1 (epidermolysis bullosa, dystrophic, dominant and recessive, COL7A1, Parente et al., 1991 ), Kunitz-type protease inhibitor 3 precursor (HKIB9, SPIT 3, Deloukas et al., 2001 ), WAP four-disulfide core domain 8 (WFDC8, Clauss et al., 2002 ), WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing (WFIKKN, 2 KD, Trexler et al., 2001 ), WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing 2 (WFIKKN2, 2 KD, Hill et al., 2003 ), as well as hypothetical proteins or open reading frames, which are used in the Analysis of the human genome were identified, such. Serine peptidase inhibitor, Kunitz type 4 (SPINT 4, Corf 137), papilin, proteoglycan-like sulfated glycoprotein (PAPLN, hypothetical protein), Q9BQP5 - OTTHUMP00000031164 (fragment, C20orf168), LOC285929, similiar to matrilin 2 precursor (hypothetical protein ).

For some of the Kunitz domains from the genes mentioned above it has been shown that they inhibit serine proteases with high potency (Nexin-II: Dennis and Lazarus, 1994 . Dennis et al., 1995 . Wagner et al., 1992 . Navaneetham et al., 2005 . Van Nostrand et al., 1990 ; Locker-1: Kirchhofer et al., 2003 ; HAI-1: Shimomura et al., 1997 . Denda et al., 2002 ; COL6A3: Kohlfeldt et al., 1996 ; WFIKKN: Nagy et al., 2003 ), but others are not further characterized in terms of their inhibitory activity spectrum. The aim was therefore to express the Kunitz domains isolated via PCR from human genomic DNA in suitable expression systems, to test them for their inhibitory effect and to prepare variants which have properties comparable to aprotinin.

Summary of the invention

aim The present invention is the recombinant production of Kunitz domains isolated from human genomic DNA and have a similar effect to aprotinin. The desired properties were determined by PCR and mutagenesis techniques achieved, the mutagenesis leading to amino acid residues, those in natural or non-natural Kunitz domains can happen.

• • die in geeigneten Hefe- und weiteren Expressionssystemen rekombinant hergestellt werden können
• • die Serin-Proteasen inhibieren können (Tabelle 1)
• • deren Plasmin-, Plasmakallikrein- und Faktor XIa-Inhibition mit Aprotinin vergleichbar ist (Tabelle 1)
• • die in funktionellen in vitro Assays zu Aprotinin vergleichbare Effekte auf Fibrinolyse und Koagulation zeigen

This approach leads to variants of human Kunitz domains

• • which can be produced recombinantly in suitable yeast and other expression systems
• • capable of inhibiting serine proteases (Table 1)
• • whose plasmin, plasma kallikrein and factor XIa inhibition are comparable to aprotinin (Table 1)
• • show comparable effects on fibrinolysis and coagulation in functional in vitro assays on aprotinin

Detailed description the invention

Kunitz domains for the purposes of this invention are homologs of aprotinin having 55 to 62 amino acid residues, usually containing six cysteine residues and three disulfide bridges, each between the positions Cys 5-Cys 55, Cys 14-Cys 38 and Cys 30-Cys 51 (Numbering according to aprotinin, see Seq. A). In addition, up to three further arbitrary amino acids can be added at the N- or / and C-terminus. 1 shows the structure of a Kunitz domain. The amino acids of a Kunitz domain are numbered so that the six cysteine residues are in positions 5, 14, 30, 38, 51 and 55. Similarly, the fourth amino acid is designated N-terminal of Cys 5 number 1. If additional amino acids are added at the N-terminus, they are given the numbers -1, -2, -3. Certain amino acids found in previously known Kunitz domains are known in U.S. Pat 1 designated accordingly. All unspecified positions may contain any amino acids.

1 shows the definition of a Kunitz domain.

aprotinin, also called bovine pancreatic trypsin inhibitor (BPTI) becomes a prototype of the Kunitz domains (Fritz and Wunderer, 1983). It is a basic protein of 58 aa Length, which is typical for Kunitz domains Contains cysteine residues at positions 5, 14, 30, 38, 51 and 55. The cysteine residues form three disulfide bridges (Cys 5 - Cys 55; Cys 14 - Cys 38; Cys 30 - Cys 51), which is the Stabilize molecule. The numbering of the amino acids the Kunitz domains according to this invention corresponds the numbering of aprotinin (see Seq. A). It is located Amino acid 1 four positions N-terminal of Cys 5 and corresponds the amino acid Arg 1 of aprotinin. Be at the N-terminus added additional amino acids obtained they are the numbers -1, -2, -3.

Seq. A: aprotinin

Kunitz inhibitors have been found in various vertebrates as well as invertebrates ( Laskowski and Kato, 1980 ). These naturally occurring Kunitz domains are referred to in this application as "Natural Kunitz domains" ( Shimomura et al., 1997 . Li et al., 1998 . Ponte et al., 1988 . Richardson et al., 2001 . Wun et al., 1988 . Marlor et al., 1997 . Petersen et al., 1994 . Vetr and Gebhard, 1990 . Chun et al., 1990 . Parente et al., 1991 . Norris et al., 1993 . Claus et al., 2002 . Trexler et al., 2001 , 2002), Kunitz domains modified by genetic engineering techniques, e.g. B. by replacement of amino acid residues and / or deletions and so do not occur in nature, are referred to in this application as non-natural Kunitz domains " US 6010880 . US 5795865 . WO 92/06111 . US 5777568 . US 6482798 . P 0 238993 . EP 0 307592 EP 0 821 007 . US 5863893 . US 5914315 . US 7019123 ).

Aprotinin is a potent inhibitor of plasmin, plasma kallikrein and factor XIa, the clinical effect of which is attributed to the inhibition of fibrinolysis, the inhibition of intrinsic blood coagulation (contact activation) and the reduction of thrombin formation ( Blauhut et al., 1991 . Dietrich et al., 1995 ).

As a bovine protein, aprotinin in humans leads to the formation of antibodies. Repeated Trasylol may cause allergic reactions (anaphylactic shock), limiting the possibility of multiple use of aprotinin ( Dietrich et al., 2001 . Beierlein et al., 2005 ).

aim Therefore, it is the Kunitz domains of the present invention of human origin or variants of these Kunitz domains to identify a comparable or improved to aprotinin possess functional activity. invention Kunitz domains inhibit both plasmin and plasma kallikrein and factor XIa with high potency. They are as well potent inhibitors of fibrinolysis and intrinsic coagulation.

invention Kunitz domains and their variants inhibit plasmin with a IC50 <2 μM, better however with an IC50 <100 nM. Plasmakallikrein is of inventive Kunitz domains and their variants with an IC50 <2 μM, better, however, with an IC50 <100 nM inhibited. Furthermore, Kunitz domains according to the invention inhibit and their variants factor XIa with an IC50 <2 μM, but better with one IC50 <100 nM.

The following are examples of human Kunitz domains according to the invention with an effect comparable or improved to aprotinin (see Table 1):
Seq. No. 38 (APP4)
Seq. No. 50 (APLP-2)
Seq. No. 54 (AMBP-D2)

It has also been found that various human Kunitz domains inhibit the serine proteases plasmin, plasma kallikrein and / or factor XIa only with low potency or not at all. The following are examples of such human Kunitz domains (see Table 1):
Seq. No. 42 (TFPI1-D2)
Seq. No. 48 (TFPI2-D3)
Seq. No. 66 (WFI-2-D2)
Seq. No. 63 (WFI-1-D2)
Seq. No. 27 (COL6A3-6His)

Surprisingly was found to be through the targeted introduction of mutations the inhibitory effect of such human Kunitz domains improved with respect to plasmin, plasma kallikrein and factor XIa can be, so that the resulting variants a the Aprotinin have comparable or improved activity.

Variants of human Kunitz domains according to the invention have the following amino acids at the positions numbered according to aprotinin:
X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = HX19 = P, X20 = R, X21 = W, X22 = W.

The remaining Positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:
X10 = V, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F.

The remaining Positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:
X10 = E, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F.

The remaining Positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:
X10 = V, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F.

The remaining Positions contain any amino acids of human Kunitz domains.

Further variants according to the invention of human Kunitz domains have the following amino acids at the positions numbered according to aprotinin:
X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F.

The remaining Positions contain any amino acids of human Kunitz domains.

Particularly preferred are the following variants of human Kunitz domains:
Seq. No. 60d (COL6A3-mut4)
Seq. No. 60e (COL6A3-mut5)
Seq. No. 63a (WFI1-D2-mut1)
Seq. No. 63c (WFI1-D2-mut3)
Seq. No. 63d (WFI1-D2-mut4)

Human Kunitz domains according to the invention and their variants also inhibit fibrinolysis and intrinsic coagulation in human plasma with aprotinin comparable or improved efficacy ( 1 and 2 )

The and comparable with aprotinin properties possessed human Kunitz domains and their variants are suitable for the treatment of the following disease states: Blood loss in operations with increased risk of bleeding; Therapy of thromboembolic conditions (eg after surgery, Accidents), shock, polytrauma, sepsis, disseminated intravascular Coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, Stroke, embolism, deep vein thrombosis, inflammatory Diseases (eg, rheumatism, asthma), invasive tumor growth, and Metastasis, pain and edema therapy (brain edema, Spinal edema), prevention of activation Hemostasis in dialysis treatment, treatment of symptoms skin aging (elastosis, atrophy, wrinkling, vascular changes, Pigment changes, actinic keratosis, blackheads, cysts), Wound healing, skin cancer, treatment of skin cancer symptoms (actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant Melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, infections of the brain, Tendinopathy.

The invention relates to an isolated protein or polypeptide which contains a Kunitz domain and is characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:
X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = HX19 = P, X20 = R, X21 = W, X22 = W, the remaining positions containing any amino acids of human Kunitz domains;
or
characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:
X10 = V, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains;
or
characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:
X10 = E, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains;
or
characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:
X10 = V, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains;
or
characterized in that the domain has the following amino acids at the positions numbered according to aprotinin:
X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains.

According to the invention an isolated protein as described above, with the remainder Positions of Kunitz Domain Amino Acids One contain specific human Kunitz domain.

Especially preferred is an isolated protein or polypeptide containing an amino acid sequence selected from the group consisting of Seq. No. 60d (COL6A3-mut4, SEQ ID NO: 121), Seq. No. 60e (COL6A3-mut5, SEQ ID NO: 122), Seq. No. 63a (WFI1-D2-mut1, SEQ ID NO: 126), Seq. No. 63c (WFI1-D2-mut3, SEQ ID NO: 128) and Seq. No. 63d (WFI1-D2-mut4, SEQ ID NO: 129).

The The invention further relates to a fragment of any of the above described proteins or polypeptides, characterized that the fragment has an inhibitory effect Plasmin, Plasmakallikrein, or Factor XIa, with the Effect of aprotinin comparable or better than the effect of aprotinin is.

Also According to the invention, a nucleic acid, which for a polypeptide or protein as described above coded. The nucleic acid may be heterologous vector sequences or a heterologous promoter sequence 5 'to the coding region.

According to the invention also a cell or a non-human organism, the one above contain described nucleic acid.

The The invention also relates to a method for the isolation of an inventive Polypeptide or protein, characterized in that a cell, the one for a protein according to the invention or polypeptide-encoding nucleic acid is cultured and the protein is purified.

The Invention also relates to a monoclonal or polyclonal antibody, specific to an inventive Protein.

The The invention further relates to the use of an inventive Protein or polypeptide or a novel Nucleic acid for the manufacture of a medicament for the treatment selected from conditions or diseases the group consisting of blood loss in operations with elevated Risk of bleeding; thromboembolic states, shock, polytrauma, Sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, embolism, deep Venous thrombosis, inflammatory diseases (eg rheumatism, Asthma), invasive tumor growth and metastasis, pain and edema therapy (Cerebral edema, spinal edema), prevention the activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkling, Vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, Treatment of symptoms of skin cancer (actinic keratosis, basal cell carcinoma, Squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, Cerebral hemorrhage, inflammation of the brain and spinal cord, Infections of the brain and tendinopathy.

Especially preferred is the use of an inventive Protein or polypeptide or a novel Nucleic acid for the manufacture of a medicament for the treatment selected from conditions or diseases the group consisting of blood loss in operations with elevated Risk of bleeding; thromboembolic states, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), Myocardial infarction, stroke, embolism, deep vein thrombosis, and wound healing.

Brief description of the figures

1 shows the definition of a Kunitz domain.

2 describes the antifibrinolytic activity of WFI1-D2-mut4 in human plasma.

3 describes the anticoagulant activity of WFI1-D2-mut4 in human plasma.

embodiments

Molecular biological, cell biological and biochemical techniques

Routine cloning work was done according to Sambrook et al. ( Molecular Cloning Cold Spring Harbor, 1989 ) carried out. For the isolation of plasmid DNA from E. coli (so-called mini- and midipreps) Qiagen-tips (Qiagen) were used. As a host organism for transformations, the E. coli strain DH5α (Stratagene) was used. The extraction of DNA fragments from agarose gels was performed using the Qiagen gel extraction kit according to the manufacturer (Qiagen).

oligonucleotides for site directed mutagenesis experiments, primers for PCR and sequencing reactions were purchased from the company Operon, synthetic genes (optimized for S. cerevisiae coden-usage) of the company Geneart.

In vitro Mutageneses were site-directed using the Quick-change II XL Mutagenesis kits according to the manufacturer (Stratagene) performed. For PCR experiments, kits from Qiagen (Hot Star Mastermix), Stratagene (PfuUltra Hotstart DNA Polymerase) or Novagen (KOD HiFi, Hot Start and XL DNA Polymerases) according to the manufacturer used for the purification of PCR fragments was the PCR Purification Kit from Qiagen.

For cell-free transcription and translation experiments, the Rapid Translation System (RTS) pIVEX HIS tag, 2 ^nd Generation Vector Set was used according to the manufacturer (Roche). All vector constructs and mutagenesis were confirmed by cycle DNA sequencing with fluorescently-labeled terminators (Big Dye Terminator, Version 1.1, Applied Biosystems) on a sequencer (3100 Avant Genetic Analyzer, Applied Biosystem).

example 1

Cloning of protease inhibitor domains of the Kunitz type from human genomic DNA by PCR

• • Amyloid beta (A4) precursor Protein (peptidase nexin-II, Alzheimer disease)
  
• • Serine peptidase inhibitor-like, with Kunitz and WAP domains 1 (Eppin)
  
• • Tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor, TFPI-1), Domäne 2 und 3
  
• • Tissue factor pathway inhibitor 2 (TFPI-2 ), Domäne 2 und 3
  
• • Amyloid beta (A4) precursor-like Protein 2 (APLP 2)
  
• • Alpha-1-microglobulin/bikunin precursor (AMBP), Domäne 1 und 2
  
• • Serine peptidase inhibitor, Kunitz type 1 (SPINT 1, HAI-1), Domäne 1 und 2
  
• • Collagen, type VI, alpha 3 (COL6A3)
  
• • Collagen, type VII, alpha 1 (COL7A1)
  
• • WAP four-disulfide core domain 8 (WFDC 8, WAP 8)
  
• • WFIKKN1 WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing (WFI-1), Domäne 1 und 2 (NM_175575)
  
• • WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing (WFI-2), Domäne 1 und 2 (NM_053284)
  
• • Serine peptidase inhibitor, Kunitz type 4 (SPINT 4, C20orf137)
  
• • Papilin, proteoglycan-like sulfated glycoprotein (PAPLN, Hypothetical Protein – hypro)
  
• • Q9BQP5_HUMAN (c20orf168)
  
• • Homo sapiens similar to matrilin 2 precursor, transcript variant 2 (LOC285929)
  
• • Similar to Kunitz-type protease inhibitor 3 precursor (HKIB9, SPIT 3, P49223)
  

For cloning via PCR, corresponding PCR primers (1a, b-23a, b) were derived for the following human genes which contain protease inhibitor domains of the Kunitz type (referred to below as human Kunitz domains) in their coding sequences:

• Amyloid beta (A4) precursor protein (peptidase nexin-II, Alzheimer disease)
  
• Serine peptidase inhibitor-like, with Kunitz and WAP domains 1 (Eppin)
  
• Tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor, TFPI-1), domains 2 and 3
  
• • Tissue factor pathway inhibitor 2 (TFPI-2), domains 2 and 3
  
• Amyloid beta (A4) precursor-like protein 2 (APLP 2)
  
• Alpha-1-microglobulin / bicunin precursor (AMBP), domains 1 and 2
  
• Serine peptidase inhibitor, Kunitz type 1 (SPINT 1, HAI-1), domains 1 and 2
  
• • Collagen, type VI, alpha 3 (COL6A3)
  
• • Collagen, type VII, alpha 1 (COL7A1)
  
• WAP four-disulfide core domain 8 (WFDC 8, WAP 8)
  
• • WFIKKN1 WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing (WFI-1), domains 1 and 2 (NM_175575)
  
• WAP, follistatin / kazal, immunoglobulin, kunitz and netrin domain containing (WFI-2), domains 1 and 2 (NM_053284)
  
• Serine peptidase inhibitor, Kunitz type 4 (SPINT 4, C20orf137)
  
• • Papilin, proteoglycan-like sulfated glycoprotein (PAPLN, Hypothetical Protein - hypro)
  
• • Q9BQP5_HUMAN (c20orf168)
  
• Homo sapiens similar to matrilin 2 precursor, transcript variant 2 (LOC285929)
  
• • Similar to Kunitz-type protease inhibitor 3 precursor (HKIB9, SPIT 3, P49223)
  

The PCR reactions each contained about 100 ng of human genomic DNA, 10 pMol gene-specific primer-up, 10 pmol gene-specific primer-down (dwn), 1 mM dNTPs, 1xPCR reaction buffer (Stratagene), 2.5 U PfuUltrahotstart DNA polymerase (Stratagene) in a total volume of 50 μl.

The Cycle conditions were 5 min. at 94 ° C, 35 cycles with each 1 min. at 94 ° C, 1 min. at 50 ° C, 1 min. at 72 ° C and a subsequent incubation for 10 min at 72 ° C. The individual PCR reactions were with purified in a purification kit (Qiagen) into the plasmid vector subcloned pCR-blunt (invitrogen) and each sequence checked by cycle DNA sequencing and confirmed.

The Kunitz-type protease inhibitor domains amplified by PCR from human genomic DNA had the following deduced amino acid sequences:

Example 2

Cloning of human Kunitz domains in the veckor pIVEX2.3d

For cell-free transcription and tranlation experiments, human Kunitz domains were cloned into the vector pIVEX2.3d by PCR and appropriate restriction enzymes. By way of example, the cloning of the human Kunitz domain APP4 (amyloid beta-A4 precursor protein, peptidase nexin-II) is shown:

accompanying and restriction sites comprising sequences (24a: NcoI: ccatgg, 24b: XmaI: cccggg) are shown in lowercase letters, Recognition sequences for restriction enzymes are underlined, human Kunitz domain coding sequences are shown in capital letters. The PCR was performed with a Purification Kit (Qiagen) purified with the restriction enzymes NcoI and XmaI cut and in which also with NcoI and XmaI ligated vector pIVEX2.3d ligated.

With The ligation mixture was transformed into E. coli DH5α cells. The resulting clone (designated APP4.pIVEX) became the sequence determined by DNA sequence analysis.

The coding region of the APP4.pIVEX clone comprises 212 bp (scanned from 2 stop codons), encodes a protein (APP4.pep) of 71 amino acids in total and has the following sequence:

Amino acids (aa) 1 and 2 at the N-terminus are from the vector construct, aa 3-60 represent the human Kunitz domain, aa 61-71 are left and one histidine part (6xHis). The protein APP4 was used in a cell-free transcription and translation system (RTS, Roche) and successfully produced a protein of the expected size (8 kDa). Analogously to the example APP4 described above, the following human Kunitz domains were each cloned into the vector pIVEX2.3d:

Example 3

Cloning of human Kunitz domains in yeast secretion vectors pIU10.10.W and p1U3.12.M

The commercially available E. coli / S. cerevisiae shuttle vector pYES2 (Invitrogen) was modified and served as starting material for the construction of the yeast secretory vectors pIU10.10.W and pIU3.12.M (Apeler, 2005).

through PCR and appropriate primers with appropriate restriction sites the human Kunitz domains were prepared from the vector pIVEX2.3d into the yeast secretion vectors pIU10.10.W and pIU3.12.M recloned.

exemplary is the cloning of the human Kunitz domain APP4 (amyloid beta-A4 precursor protein, peptidase nexin-II) shown.

The Cloning into the yeast secretory vector pIU10.10.W was carried out via the restriction sites BsaBI (GAT tccc ATC in primer 25a), and XhoI (CTCGAG in primer 25b), for cloning in pIU3.12.M, the restriction sites HindIII (AAGCTT in primer 26a) and BamHI (GGATCC in primer 26b). used as a template in the respective PCR reactions Seq. Number 1.

The primers had the following sequences:

The PCR reactions were purified with a purification kit (Qiagen), with the restriction enzymes BsaBI and XhoI for cloning in pIU10.10.W or with HindiIII and BamHI for cloning cut into pIU3.12.M and ligated into the appropriate vectors.

With The ligation approaches were E. coli DH5α cells transformed and from the resulting clones by the DNA sequence Sequence analysis determined.

Derived amino acid sequence of the human Kunitz domain APP4 cloned into yeast secretion vector pIU10.10.W (the pre-sequence of MFα mating factor α - is underlined, the amino acid sequence of APP4 is in bold):

Derived amino acid sequence of the human Kunitz domain APP4 cloned into yeast secretion vector pIU3.12.M (the pre-sequence of the MFα mating factor α - is underlined, the MFα-Pro sequence is italic, the amino acid sequence of the human Kunitz domain APP4 is in bold): Seq. No. 38, SEQ ID NO: 95:

Both Expression constructs (APP4 in yeast secretory vector pIU10.10.W and pIU3.12.M) were successfully expressed in baker's yeast.

Analogous as shown by the example of the human Kunitz domain APP4, other human Kunitz domains were inserted into the yeast secretion vectors pIU10.10.W and pIU3.12.M cloned. All constructs in the yeast secretion vector pIU10.10.W contain each of the pre-sequence of the Mating Factor α (MFα) before the for human Kunitz domain coding sequence (see SEQ ID NO 37), Constructs in the yeast secretory vector pIU3.12.M contain each the pre-pro sequence of the mating factor α (see seq. 38). As synthetic genes Seq. No. 63a-Seq. No. 63d and Seq. No. 64a-Seq. No. 64d (mutated sequences are underlined).

The deduced amino acid sequences are as follows:

Example 4

Transformation of Saccharomyces cerevisiae

Yeast cells, e.g. B. strain JC34.4D (MATα, ura3-52, suc2) were grown in 10 ml of YEPD (2% glucose, 2% peptone, 1% Difco yeast extract) and harvested at an OD ₆₀₀ of 0.6 to 0.8 , The cells were washed with 5 ml of solution A (1 M sorbitol, 10 mM bicin pH 8.35, 3% ethylene glycol), resuspended in 0.2 ml of solution A and stored at -70 ° C.

plasmid DNA (5 μg) and carrier DNA (50 μg of herring sperm DNA) were given to the frozen cells. The cells were subsequently thawed by shaking for 5 min at 37 ° C. After adding 1.5 ml of solution B (40% PEG 1000, 200 mM bicine pH 8.35), the cells were kept at 30 ° C for 60 min after pelleting with 1.5 ml of solution C (0.15 M NaCl; 10 mM Bicin pH 8.35) and in 100 μl of solution C resuspended. The Ausplattierung took place on a selection medium with 2% agar. Transformants were after an incubation of 3 days at 30 ° C.

Example 5

Fermentation of the yeast cells

nutrient solutions

• (Ad 1000 ml in H₂O dest. lösen, pH-Wert mit NaOH auf 3–4 einstellen. Charge in max. 12 Gebinde aufteilen und bei –18°C lagern; die einzelnen Gebinde aufgetaut max. 1 Monat einsetzen.)

For fermentation of yeast cells for the expression of aprotinin or KTPI variants with modified amino acid sequence, the following nutrient solutions were used broth feedstock SD2 SC5 Bacto-Yeast Nitrogen Base 6.7 g / l - Difco Bacto-Yeast Extract - 20.0 g / l glucose 20.0 g / l 20.0 g / l KH2PO4 6.7 g / l 6.7 g / l (NH4) 2SO4 - 2.0 g / l MgSO4 × 7H2O - 1.0 g / l Trace element solution 4 - 1.0 ml / l pH after NaOH-Titr. 6 6 SL4 solution: Titriplex III (Merck 8418) 5 g FeSO ₄ .7H ₂ O (Merck 3965) 2 g ZnSO ₄ .7H ₂ O (Merck 8883) 0.1 g MnCl ₂ .4H ₂ 0 (Merck 5927) 30 mg H ₃ BO ₃ (Merck 165) 0.3 g CoCl ₂ .6H ₂ O (Merck 2533) 0.2 g CuCl ₂ .2H ₂ O (Merck 2733) 10 mg NiCl ₂ .6H ₂ O (Merck 6717) 20 mg Na ₂ MoO ₄ .2H ₂ O (Merck 6521) 30 mg

• (Dissolve 1000 ml in distilled H ₂ O, adjust the pH to 3-4 with NaOH, divide the batch into a maximum of 12 containers and store at -18 ° C, thawing the individual containers for a maximum of 1 month.)

The Feedstocks were prepared in demineralized water and the pH adjusted to pH 5.5. Sterilization was carried out for 20 min at 121 ° C. Glucose was required in 1/5 of the required Volume dissolved in demineralized water, sterilized separately and after cooling to the remaining nutrient solution where.

strain stocks

 strain stocks All yeast transformants were prepared by mixing 1 ml aliquots a preculture with 1 ml of 80% glycerol solution and storage created at -140 ° C.

precultures

The Pre-culture fermentations were carried out in 50 ml (for main cultures in small volume) or 1 liter shake flask (for Main cultures in the middle volume), filled with 10 resp. 100 ml of SD2 nutrient solution. The Inoculation took place with a trunk preserve or with a single colony from an SD2 agar plate. The cultures were under constant Shaking (240 rpm) for 2-3 days Incubated at 28-30 ° C.

Main culture fermentations

The Main culture fermentations on a small scale took place under Use of 1 liter shake flasks filled with 100 ml SC5 nutrient solution. The inoculation took place usually with 3 ml of the preculture described above. Subsequently The cultures were under constant shaking (240 rpm) for 4 days at 28-30 ° C incubated.

In the case of the main culture fermentation on a medium or large scale, the bioreactor system of Wave Biotech (Tagelswangen, CH) was used. Specifically, 1000 or 10000 ml of SC5 medium were inoculated with 30 or 300 ml preculture and incubated for 4 days at 30 ° C in the Wavebag at a rocking frequency of 32 / min (angle: 10 °, air supply: 0.25 ltr. / min). The pH of the cultures was checked on days 1 to 3 and adjusted as necessary with 5 N NaOH to pH 5-6. The cultures were fed on days 1 to 3, with 1 ml each of 50% yeast extract solution and 4 ml of 4 M glucose solution in the case of 100 ml cultures. In the case of the 1000 and 10000 ml cultures, the addition of the 10 or 100 times the volume of feed solutions was carried out continuously during the day. For growth control, the OD _{600 of} the cultures could be determined at different times.

To 4 days was the harvest of cell-free supernatants by centrifugation (15 min at 6000 rpm in JA14 rotor).

Example 6

Purification of human Kunitz domains from supernatants of fermented yeast cells

The human Kunitz domain produced in the main culture fermentation with the Seq. No. 35 containing cell-free supernatants were added with 1 M NaOH until the pH was 7.8. Suspended particulates suspended in the supernatant were sedimented by centrifugation at 2,000 rpm at 4 ° C (15 minutes, Beckman-Allegra 6KR). The supernatant was applied to a 10 ml trypsin agarose column (Sigma-T1763) at 1 ml / min. applied. The column was then washed with 70 ml of 50 mM Tris pH 7.8, 250 mM NaCl and 50 ml of 50 mM Tris pH 7.8, 600 mM NaCl. It was then eluted with 180 ml of 50 mM KCl / 10 mM HCl pH 2.0 and with 100 ml of 50 mM KCl / 250 mM HCl pH 0.9. The 2 ml fractions were collected in tubes containing 500 μl each of 200 mM Tris pH 7.6, 2 M NaCl to neutralize. Human Kunitz domains containing Sequence No. 35 were identified by the trypsin inhibition assay described below. Trypsin-inhibiting fractions were pooled, mixed with the same volume of 0.1% TFA and applied to a Source 15 RPC column (GE Healthcare). The column was washed with 6 ml of 0.1% TFA (buffer HPLC-A) and then the human Kunitz domain was digested with Seq. No. 35 with a 25 ml gradient on 50% buffer HPLC-B (0.1% TFA, 60% acetonitrile) and another 5 ml gradient on 100% buffer HPLC-B eluted. The Seq. No. 35 were lyophilized and the lyophilizate was taken up in 250 μl of 50 mM Tris pH 7.5 per fraction.

Example 7

Production of Human Kunitz Domains by in vitro translation and purification via Ni-NTA agarose

The Kunitz domain with the Seq. No. 27e was used of the plasmid pIVEX2.3d by means of in vitro translation with the RTS-500 E. coli disulfide kit (Roche Diagnostics) according to the regulations expressed by the manufacturer.

To At the end of expression, both solutions (reaction solution and supply solution) and mixed with 1 ml of 0.1% Tween-20 added. Insolubles were removed by centrifugation at 3,000 rpm (4 ° C, 30 min) away. Subsequently was the clear supernatant with 300 ul Ni-NTA Superflow (Qiagen) mixed and the resulting suspension under constant Agitation incubated at 4 ° C for 90 min. The affinity matrix was sedimented by centrifugation and the supernatant discarded. Ni-NTA Superflow was incubated three times by incubation Washed 1 ml of 50 mM Tris pH 7.5, 100 mM NaCl, 10 mM imidazole and each sedimented by centrifugation. Subsequently became the Ni-NTA Superflow bound Kunitz domain with the Seq. No. 27e by three times incubation of the affinity matrix with 900 μl each of 50 mM Tris pH 7.5, 100 mM NaCl, 250 mM imidazole eluted. The eluates were pooled and desalted with NAP-25 columns (GE Healthcare) buffered to 10 mM Tris pH 7.5. After lyophilization became the Kunitz domain with the Seq. No. 27e in water taken up and stored at -80 ° C.

Example 8

Characterization of APP4 on the basis of tryptic peptides and molecular weight

to Molecular weight determination of APP4 became the purified protein with a 0.1% formic acid solution to 2 pmol / μl diluted and simultaneously acidified. This sample was after separation via a GromSil 120 ODS-4 HE (3 μm, 250 × 0.2 mm) was analyzed by mass spectrometry. Based Of the multiply charged molecular ions could the molecular weight be detected by APP4.

to more accurate determination of the amino acid sequence was the protein after denaturation with guanidinium hydrochloride with dithiothreitol reduced, derivatized by means of iodoacetamide and then tryptically split. The resulting split peptides were then mass spectrometry analyzed and using the proven peptide masses and MS / MS spectra the sequence coverage of APP4 detected. Here was the entire Amino acid sequence can be detected.

Example 9

Determination of inhibitory potency of human KTPI muteins against trypsin, plasmin, FXIa and plasma kallikrein

The inhibitory potency of human KTPI muteins against the enzymatic activities of trypsin, plasmin, FXIa and plasma kallikrein were determined in biochemical assays in white 384-well microtiter plates using fluorogenic substrates. The assay buffer was composed of 50mM Tris / Cl, pH 7.4, 100mM NaCl, 5mM CaCl ₂ , 0.08% (w / v) BSA. The test conditions were as follows: enzyme Enzmyendkonz. Order No. substratum Substratendkonz. Order No. trypsin 5 ng / ml T-6424 (Sigma) Boc-Ile-Glu-Gly-Arg-AMC 5 μM I-1100 (Bachem) plasmin 50 ng / ml Hplas (Enzyme Research) MeOSuc-Ala-Phe-Lys-AMC 50 μM I-1275 (Bachem) FXIa 462 nM HFXIa 1111a-1 (Enzyme Research) Boc-Glu (OBzl) -Pro-Arg-AMC 5 μM I-1575 (Bachem) Plasma kallikrein 1nM 420307 (Calbiochem) Boc-Val-Pro-Arg-AMC 80 μM ES-011 (R & D)

Per hole, 10 μl of a serial dilution were initially charged and preincubated with 20 μl of enzyme for 5 min at RT. Subsequently, the reaction was started by addition of 20 .mu.l of substrate. The measurement was carried out after 60-90 min in a Tecan reader at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Dose-response curves and half-maximal inhibitory constants (IC50 values) were determined using GraphPad Prism software (version 4.02). determined. Table 1: IC50 values of some Kunitz domains for the inhibition of human trypsin, plasmin, factor XIa and plasma kallikrein Kunitz domain Trypsin IC50 [nM] Plasmin IC50 [nM] Factor XIa IC50 [nM] Plasma kalli-krein IC50 [nM] Seq. A (aprotinin, BPTI) 11 3 393 41 Seq. No. 38 (APP4) <1 299 <1 41 Seq. No. 42 (TFPI-1-D2) <1 2266 2578 3714 Seq. No. 36 (TFPI-2-D3) > 10000 > 10000 > 10000 > 10000 Seq. No. 50 (APLP-2) <1 227 28 117 Seq. No. 54 (AMBP-D2) 12 692 189 377 Seq. No. 66 (WFI-2-D2) 24 > 10000 1696 827 Seq. No. 63 (WFI-1-D2) 54 1042 > 3000 > 3000 Seq. No.63a (WFI-1-D2-mut1) 70 475 976 149 Seq. No. 63d (WFI-1-D2-mut4) <1 11 85 5 Seq. No. 63c (WFI-1-D2-mut3) <1 12 680 152 Seq. No. 27 (Col6A3) > 10000 > 10000 > 10000 > 10000 Seq. No. 27a (Col6A3-mut1) > 10000 > 10000 > 10000 > 10000 Seq. No. 27b (Col6A3-mut2) > 10000 > 10000 > 10000 > 10000 Seq. No. 60d (Col6A3-mut4) 2 229 1919 287 Seq. No. 60e (Col6A3-mut5) 30 107 371 23

Example 10

Inhibition of fibrinolysis by Seq. No. 63d (WFI-1-D2-mut-4)

The effect of WFI-1-D2-mut-4 was tested in an in vitro fibrinolytic model and compared to the effect of trasylol (aprotinin). Human citrated plasma was spiked with 0.13 pM tissue factor (TF) and 164 U / ml tissue plasminogen activator (tPA) and WFI-1-D2 mut-4 or aprotinin at various concentrations (0.1 μM to 10 μM) and 37 min at 37 min ° C incubated. As a control, physiological saline was used in place of the Kunitz domains. Clot formation by TF and clot lysis by tPA were determined by optical density measurements (OD ₄₀₅ nm) with a Tecan Safire. Fibrinolysis was defined as the relative decrease in OD after clot formation. Inhibition of the relative decrease in OD is the measure of antifibrinolytic activity. WFI-1-D2-mut-4 inhibited fibrinolysis with an IC ₅₀ of 2.0 ± 0.2 μM. Trasylol (aprotinin) inhibited fibrinolysis in this model with an IC ₅₀ of 1.2 ± 0.2 μM. 2 describes the antifibrinolytic activity of WFI1-D2-mut4 in human plasma.

Example 11

Inhibition of intrinsic coagulation by Seq. No. 63d (WFI-1-D2-mut-4)

The effect of WFI-1-D2-mut-4 was tested in an in vitro coagulation model and compared to the effect of trasylol (aprotinin). Human citrated plasma was spiked with 12 mM CaCl ₂ to induce coagulation and with WFI-1-D2-mut-4 or aprotinin in various concentrations (0.1 μM to 30 μM). As a control, physiological saline was used in place of the Kunitz domains. During incubation for 90 min at 37 ° C, the increase in OD at 405 nm was determined as a measure of coagulation. From this, the half-maximal coagulation time was calculated. An extension of the half-maximal coagulation time means inhibition of coagulation. WFI-1-D2-mut-4 inhibited coagulation with a CT2 (doubling the half-maximal coagulation time) of 0.8 ± 0.02 μM. The CT2 for trasylol (aprotinin) was 12 ± 0.2 μM

3 describes the anticoagulant activity of WFI1-D2-mut4 in human plasma.

literature

• Apeler et al., 2004. Drug Discovery 54, 483-497
• Apeler in: J. Knäblein (Ed.), Modern Biopharmaceuticals, Wiley-VHC, Weinheim, 2005, pp.1021-1032
• Beierlein et al., 2005, Ann. Thorac. Surg. 79, 741-748
• Blauhut et al., 1991, J. Thorac. Cardiovasc Surg 101, 958-967
• Bode and Huber, 1992, Eur. J. Biochem 204, 433-451
• Chu et al., 1988, J. Biol. Chem. 263, 18601-18606
• Chun et al., 1990, EMBO Journal 9, 385-393
• Clauss et al., 2002, Biochem Journal 368, 233-242
• Deloukas et al., 2001, Nature 414, 865-871
• Dennis et al., 1995, J. Biol. Chem. 270, 25411-25417
• Dennis and Lazerus, 1994, J. Biol. Chem., 269, 221 29-221 36
• Dietrich et al., 1995, Anesthesiology 83, 679-689
• Dietrich et al., 2001, Anesthesiology 95, 64-71
• Delaria et al., 1997, J. Biol. Chem. 272, 12209-12214
• Denda et al., 2002, J. Biol. Chem. 277, 14053-14059
• Fritz and Wunderer, 1983, Arzneimittelforschung 33, 479-494
• Hess Jr., 2005, Am. J. Health. Syst. Pharm 62 (18 Suppl 4): 6-9
• Hill et al., 2003, Mol. Endocrinol. 17, 1144-1154
• Kawaguchi et al., 1997, J. Biol. Chem. 272, 27558-27564
• Kirchhofer et al., 2003, J. Biol. Chem. 278, 36341-36349
• Kohlfeld et al., 1996, Eur. J. Biochem. 238, 333-340
• Krowarsch et al., 2005, Protein Pept. Lett. 12, 403-407
• Laskowski and Kato, 1980, Ann. Rev. Biochem 49, 593-626
• Li et al., 1998, American Journal of Alzheimer Disease, September / October, 236-244
• Markland, et al., 1996a, Biochemistry 35, 8045-8057
• Markland et al., 1996b, Biochemistry 35, 8058-8067
• Marlor et al., 1997, J. Biol. Chem. 272, 12202-12208
• Nagy et al., 2003, J. Biol. Chem. 270, 2101-2107
• Navaneetham et al., 2005, J. Biol. Chem., 280, 36165-36175
• Oltersdorf et al., 1989, Nature 341, 144-147
• Otlewski et al., 2001, Acta. Biochim. Pole. 48, 419-428
• Parente et al., 1991, Proc. Nat. Acad. Sci. 88, 6931-6935
• Petersen et al., 1994, FEBS Letters 338, 53-57
• Ponte et al., 1988, Nature 331, 525-527
• Richardson et al., 2001, Gene 270, 93-102
• Royston, 1992, J. Cardiothorac. Vasc. Anesth 6, 76-100
• Sambrook et al., 1989, Molecular Cloning Cold Spring Harbor, 2nd Edition Schechter and Berger, 1967, Biochem. Biophys. Res. Commun. 27, 157-162
• Shimomura et al., 1997, J. Biol. Chem. 272, 6370-6376
• Sprecher et al., 1994, Proc. Nat. Acad. Sci. 91, 3353-3357
• Trexler et al., 2001, Proc. Nat. Acad. Sci. 98, 3705-3709
• Trexler et al., 2002, Biol. Chem. 383, 223-228
• van der Logt et al., 1991, Biochemistry 30, 1571-1577
• Van Notstrand et al., 1990, J. Biol. Chem. 265, 9591-9594
• Vetr and Gebhard 1990, Biol. Chem. Hoppe-Seyler 371, 1185-1196
• Wagner et al., 1992, Biochem. Biophys. Rees. Commun., 186, 1138-1145
• Wun et al., 1988, J. Biol. Chem. 263, 6001-6004
• Yang et al., 1996, Genomics 35, 24-29
• US 6,010,880 A
• US 5,795,865 A
• WO92 / 06111 A1
• US Pat. No. 6,482,798 B3
• EP 0 238 993 A2
• EP 0 307 592 A2
• EP 0 821 007 A2
• US 5,863,893 A.
• US 5,914,315 A
• US 7,019,123 B2

SEQUENCE LISTING

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - US 6010880 [0014]
• US 5795865 [0014]
• WO 92/06111 [0014]
• US 5777568 [0014]
• - US 6482798 [0014]
• - EP 238993 [0014]
• EP 0307592 [0014]
• - EP 0821007 [0014]
• US 5863893 [0014]
• US 5914315 [0014]
• US 7019123 [0014]
• - US 6010880A [0091]
• US 5795865A [0091]
• WO 92/06111 A1 [0091]
• US 6482798 B2 [0091]
• EP 0238993 A2 [0091]
• EP 0307592 A2 [0091]
• EP 0821007 A2 [0091]
• US 5863893A [0091]
• US 5914315A [0091]
• US 7019123 B2 [0091]

Cited non-patent literature

• - Laskowski and Kato, 1980; Bode and Huber, 1992 [0002]
• - Schechter and Berger, 1967 [0004]
• Otlewski et al., 2001 [0004]
• Apeler et al., 2004 [0004]
• Krowarsch et al., 2005 [0004]
• - Fritz and Wunder, 1983 [0004]
• Krowarsch et al., 2005 [0004]
• - Royston, 1992 [0005]
• - Blauhut et al., 1991 [0005]
• Dietrich et al., 1995 [0005]
• Dietrich et al., 2001 [0006]
• Beierlein et al., 2005 [0006]
• - Ponte et al., 1988 [0007]
• Oltersdorf et al., 1989 [0007]
• Richardson et al., 2001 [0007]
• Delaria et al., 1997 [0007]
• - Sprecher et al., 1994 [0007]
• Wun et al., 1988 [0007]
• van der Logt et al., 1991 [0007]
• Yang et al., 1996 [0007]
• - Vetr and Gebhard, 1990 [0007]
• Shimomura et al., 1997 [0007]
• Chu et al., 1988 [0007]
• Parente et al., 1991 [0007]
• - Deloukas et al., 2001 [0007]
• - Clauss et al., 2002 [0007]
• - Trexler et al., 2001 [0007]
• Hill et al., 2003 [0007]
• - Dennis and Lazarus, 1994 [0008]
• Dennis et al., 1995 [0008]
• - Wagner et al., 1992 [0008]
• - Navaneetham et al., 2005 [0008]
• Van Nostrand et al., 1990 [0008]
• - Kirchhofer et al., 2003 [0008]
• Shimomura et al., 1997 [0008]
• - Denda et al., 2002 [0008]
• Kohlfeldt et al., 1996 [0008]
• Nagy et al., 2003 [0008]
• - Laskowski and Kato, 1980 [0014]
• Shimomura et al., 1997 [0014]
• - Li et al., 1998 [0014]
• - Ponte et al., 1988 [0014]
• Richardson et al., 2001 [0014]
• -. Wun et al, 1988 [0014]
• Marlor et al., 1997 [0014]
• Petersen et al., 1994 [0014]
• - Vetr and Gebhard, 1990 [0014]
• Chun et al., 1990 [0014]
• Parente et al., 1991 [0014]
• - Norris et al., 1993 [0014]
• - Claus et al., 2002 [0014]
• - Trexler et al, 2001 [0014].
• - Blauhut et al., 1991 [0015]
• Dietrich et al., 1995 [0015]
• Dietrich et al., 2001 [0016]
• Beierlein et al., 2005 [0016]
• - Molecular Cloning Cold Spring Harbor, 1989 [0048]
• Apeler et al., 2004. Drug Discovery 54, 483-497 [0091]
• J. Knäblein (Ed.), Modern Biopharmaceuticals, Wiley-VHC, Weinheim, 2005, pp. 1021-1032 [0091]
• Beierlein et al., 2005, Ann. Thorac. Surg. 79, 741-748 [0091]
• - Blauhut et al., 1991, J. Thorac. Cardiovasc Surg 101, 958-967 [0091]
• Bode and Huber, 1992, Eur. J. Biochem 204, 433-451 [0091]
• Chu et al., 1988, J. Biol. Chem. 263, 18601-18606 [0091]
• Chun et al., 1990, EMBO Journal 9, 385-393 [0091]
• - Clauss et al., 2002, Biochem Journal 368, 233-242 [0091]
• Deloukas et al., 2001, Nature 414, 865-871 [0091]
• Dennis et al., 1995, J. Biol. Chem. 270, 25411-25417 [0091]
• Dennis and Lazerus, 1994, J. Biol. Chem., 269, 221 29-221 36 [0091]
• Dietrich et al., 1995, Anesthesiology 83, 679-689 [0091]
• Dietrich et al., 2001, Anesthesiology 95, 64-71 [0091]
• - Delaria et al., 1997, J. Biol. Chem. 272, 12209-12214 [0091]
• Denda et al., 2002, J. Biol. Chem. 277, 14053-14059 [0091]
• Fritz and Wunderer, 1983, Arzneimittelforschung 33, 479-494 [0091]
• - Hess Jr., 2005, Am. J. Health. Syst. Pharm 62 (18 Suppl 4): 6-9 [0091]
• Hill et al., 2003, Mol. Endocrinol. 17, 1144-1154 [0091]
• Kawaguchi et al., 1997, J. Biol. Chem. 272, 27558-27564 [0091]
• - Kirchhofer et al., 2003, J. Biol. Chem. 278, 36341-36349 [0091]
• Kohlfeld et al., 1996, Eur. J. Biochem. 238, 333-340 [0091]
• Krowarsch et al., 2005, Protein Pept. Lett. 12, 403-407 [0091]
• - Laskowski and Kato, 1980, Ann. Rev. Biochem 49, 593-626 [0091]
• - Li et al., 1998, American Journal of Alzheimer's Disease, September / October, 236-244 [0091]
• Markland, et al., 1996a, Biochemistry 35, 8045-8057 [0091]
• Markland et al., 1996b, Biochemistry 35, 8058-8067 [0091]
• - Marlor et al., 1997, J. Biol. Chem. 272, 12202-12208 [0091]
• Nagy et al., 2003, J. Biol. Chem. 270, 2101-2107 [0091]
• - Navaneetham et al., 2005, J. Biol. Chem., 280, 36165-36175 [0091]
• Oltersdorf et al., 1989, Nature 341, 144-147 [0091]
• - Otlewski et al., 2001, Acta. Biochim. Pole. 48, 419-428 [0091]
• Parente et al., 1991, Proc. Nat. Acad. Sci. 88, 6931-6935 [0091]
• Petersen et al., 1994, FEBS Letters 338, 53-57 [0091]
• - Ponte et al., 1988, Nature 331, 525-527 [0091]
• Richardson et al., 2001, Gene 270, 93-102 [0091]
• - Royston, 1992, J. Cardiothorac. Vasc. Anesth 6, 76-100 [0091]
• Sambrook et al., 1989, Molecular Cloning Cold Spring Harbor, 2nd Edition Schechter and Berger, 1967, Biochem. Biophys. Res. Commun. 27, 157-162 [0091]
• - Shimomura et al., 1997, J. Biol. Chem. 272, 6370-6376 [0091]
• Sprecher et al., 1994, Proc. Nat. Acad. Sci. 91, 3353-3357 [0091]
• Trexler et al., 2001, Proc. Nat. Acad. Sci. 98, 3705-3709 [0091]
• - Trexler et al., 2002, Biol. Chem. 383, 223-228 [0091]
• van der Logt et al., 1991, Biochemistry 30, 1571-1577 [0091]
• Van Notstrand et al., 1990, J. Biol. Chem. 265, 9591-9594 [0091]
• Chem. Hoppe-Seyler 371, 1185-1196 [0091]
• Wagner et al., 1992, Biochem. Biophys. Rees. Commun., 186, 1138-1145 [0091]
• Wun et al., 1988, J. Biol. Chem. 263, 6001-6004 [0091]
• Yang et al., 1996, Genomics 35, 24-29 [0091]

Claims (21)

An isolated protein or polypeptide containing a Kunitz domain, characterized in that the domain has the following amino acids at positions numbered according to aprotinin: X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = HX19 = P, X20 = R, X21 = W, X22 = W, the remaining positions containing any amino acids of human Kunitz domains; or in that the domain has the following amino acids at the positions numbered according to protinine: X10 = V, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains; or in that the domain has the following amino acids at the positions numbered according toprotinin: X10 = E, X11 = T, X15 = R or K, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, the remaining positions containing any amino acids of human Kunitz domains; or in that the domain has, at the positions numbered according to protinine, the following amino acids: X10 = V, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, wherein the remaining positions contain any amino acids of human Kunitz domains; or in that the domain has the following amino acids at positions numbered according to protinine: X10 = E, X11 = T, X15 = R, X16 = A, X17 = A, X18 = M, X19 = K, X20 = R, X21 = F, where the remaining positions contain any amino acids of human Kunitz domains. An isolated protein according to claim 1, with the remaining positions of the Kunitz domain Amino acids of a specific human Kunitz domain contain. An isolated protein or polypeptide containing an amino acid sequence selected from the group consisting from Seq. No. 60d (COL6A3-mut4, SEQ ID NO: 121), Seq. No. 60e (COL6A3-mut5, SEQ ID NO: 122), Seq. No. 63a (WFI1-D2-mut1, SEQ ID NO: 126), Seq. No. 63c (WFI1-D2-mut3, SEQ ID NO: 128), Seq. No. 63d (WFI1-D2-mut4, SEQ ID NO: 129) and Seq. No. 64e (WFI1-D2-mut5-pIU3.12.M, SEQ ID NO: 146). A fragment of a protein or polypeptide according to claims 1-3, characterized in that the fragment is an inhibitory Action against plasmin, plasma kallikrein, or factor XIa comparable to the action of aprotinin or better than the effect of aprotinin. A nucleic acid which is suitable for a Polypeptide or protein according to claims 1-4 encoded. The nucleic acid according to claim 5 containing heterologous vector sequences or a heterologous promoter sequence 5 'to the coding region. Cell, a nucleic acid according to claim Containing 6. Non-human organism, a nucleic acid according to claim 6 containing. A method for isolating a polypeptide or Protein according to claims 1-4, characterized in that a cell according to claim 7 is cultivated and the protein is purified. A monoclonal or polyclonal antibody, specific for a protein according to claims 1-4. A protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 as a medicine. A pharmaceutical composition, a protein according to claims 1-4 or a nucleic acid according to claim Containing 5. A protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 as a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss at Surgery with increased risk of bleeding; thromboembolic Conditions, shock, polytrauma, sepsis, disseminated intravascular Coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, Embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, Pain and edema therapy (brain edema, spinal edema), Prevention of activation of hemostasis in dialysis treatment, Treatment of skin aging symptoms (elastosis, atrophy, wrinkling, Vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, Treatment of symptoms of skin cancer (actinic keratosis, basal cell carcinoma, Squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, Cerebral hemorrhage, inflammation of the brain and spinal cord, Infections of the brain and tendinopathy. A protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 as a medicament for the treatment of conditions or diseases selected from the group consisting of blood loss at Surgery with increased risk of bleeding; thromboembolic Conditions, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), heart attack, stroke, embolism, deep vein thrombosis, and wound healing. Use of a protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 for the manufacture of a medicament for the treatment of conditions or diseases selected from the group consisting of Blood loss in operations with increased risk of bleeding; thromboembolic states, shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, embolism, deep venous thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive Tumor growth and metastasis, pain and edema therapy (brain edema, Spinal edema), prevention of activation Hemostasis in dialysis treatment, treatment of symptoms skin aging (elastosis, atrophy, wrinkling, vascular changes, Pigment changes, actinic keratosis, blackheads, cysts), Wound healing, skin cancer, treatment of skin cancer symptoms (actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant Melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, infections of the brain and tendinopathy. Use of a protein or polypeptide according to claims 1-4 or a nucleic acid according to claim 5 for the manufacture of a medicament for the treatment of conditions or diseases selected from the group consisting of Blood loss in operations with increased risk of bleeding; thromboembolic states, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), myocardial infarction, Stroke, embolism, deep vein thrombosis, and wound healing. A protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins as a medicine. A pharmaceutical composition comprising a protein containing a polypeptide selected from Group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins. A protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins as a drug for the treatment of conditions or disorders selected from the group consisting of blood loss in operations with elevated Risk of bleeding; thromboembolic states, shock, polytrauma, Sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, heart attack, stroke, embolism, deep Venous thrombosis, inflammatory diseases (eg rheumatism, Asthma), invasive tumor growth and metastasis, pain and edema therapy (Cerebral edema, spinal edema), prevention the activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkling, Vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, Treatment of symptoms of skin cancer (actinic keratosis, basal cell carcinoma, Squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, Cerebral hemorrhage, inflammation of the brain and spinal cord, Infections of the brain and tendinopathy. A pharmaceutical composition comprising a protein containing a polypeptide selects from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins for the treatment of conditions or diseases selected from the group consisting of blood loss in operations with increased risk of bleeding; thromboembolic conditions, shock, polytrauma, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), unstable angina, myocardial infarction, stroke, embolism, deep vein thrombosis, inflammatory diseases (eg rheumatism, asthma), invasive tumor growth and metastasis, Pain and edema therapy (cerebral edema, spinal cord edema), prevention of activation of hemostasis in dialysis treatment, treatment of skin aging symptoms (elastosis, atrophy, wrinkles, vascular changes, pigment changes, actinic keratosis, blackheads, cysts), wound healing, skin cancer, treatment of symptoms of Skin cancer (actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma), multiple sclerosis, fibrosis, cerebral hemorrhage, inflammation of the brain and spinal cord, brain infections and tendinopathy. A protein containing a polypeptide selected from the group consisting of Seq. No. 38 (SEQ ID NO: 95, APP4), Seq. No. 50 (SEQ ID NO: 107, APLP-2), and Seq. No. 54 (SEQ ID NO: AMBP-D2) or a nucleic acid encoding such proteins as a drug for the treatment of conditions or disorders selected from the group consisting of blood loss in operations with elevated Risk of bleeding; thromboembolic states, shock, sepsis, disseminated intravascular coagulation (DIC), multiple organ failure (MOF), Myocardial infarction, stroke, embolism, deep vein thrombosis, and wound healing.