WO2008110301A1 - Aprotinin variants with improved properties - Google Patents

Aprotinin variants with improved properties Download PDF

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Publication number
WO2008110301A1
WO2008110301A1 PCT/EP2008/001820 EP2008001820W WO2008110301A1 WO 2008110301 A1 WO2008110301 A1 WO 2008110301A1 EP 2008001820 W EP2008001820 W EP 2008001820W WO 2008110301 A1 WO2008110301 A1 WO 2008110301A1
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aprotinin
seq
bpti
amino acid
inhibition
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PCT/EP2008/001820
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German (de)
French (fr)
Inventor
Axel Harrenga
Felix Oehme
Heiner Apeler
Frank Dittmer
Jürgen Franz
Michael Sperzel
Beatrix Stelte-Ludwig
Karl Ziegelbauer
Simone Greven
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Bayer Schering Pharma Aktiengesellschaft
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Publication of WO2008110301A1 publication Critical patent/WO2008110301A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors
    • C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)

Definitions

  • the present invention relates to aprotinin variants with improved immunological and enzyme-inhibiting properties and their preparation and use.
  • Kunitz domains are polypeptides 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 amino acid residues long loop in the N-terminal region of the
  • Kunitz-domain This loop binds to the catalytic center of the protease and thus prevents cleavage of the corresponding protease substrates (Laskowski and Kato [1980], Bode and Huber [1992]).
  • Aprotinin also referred to as bovine pancreatic trypsin inhibitor (BPTI) (BPTI; Figure 1: SEQ ID NO: 1), is considered a prototype of the Kunitz domains (Fritz and Wunderer [1983]). It is a basic protein of 58 amino acid residues in length that can be isolated from various bovine organs (including pancreas, lung, liver and heart). Aprotinin is stabilized by three disulfide bridges (Cys 5 - Cys 55, Cys 14 - Cys 38, Cys 30 - Cys 51) and is among others. a potent inhibitor of trypsin, plasmin and plasma kallikrein.
  • BPTI bovine pancreatic trypsin inhibitor
  • X-ray diffraction analysis of the aprotinin-bovine trypsin complex revealed that the aprotinin contact region to the protease catalytic center is essentially formed by a loop consisting of amino acid residues 11 to 19 (see Bode and Huber [1992] and references therein).
  • the amino acid residue Lys 15 which is in particularly close contact with the catalytically active serine residue of the protease.
  • the amino acid residue Lys 15 is therefore designated by definition as Pl residue (Schechter and Berger [1967].) N-terminal of Lys-15 are the residues P2, P3, etc., while the amino acid residues are C-terminal of Lys-15 as Pl ', 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].) The amino acid residues 36-39 are also important for the effectiveness of aprotinin (Fritz and Wunder [1983], Krowarsch et al. [2005]).
  • aprotinin is sold under the trade name Trasylol is currently used mainly in cardiac surgery, after clinical studies have shown that treatment with aprotinin significantly reduces the need for transfusion in such operations and reduces rebleeding (Royston [1992]) It is attributed to the inhibition of intrinsic blood coagulation (contact activation), the inhibition of fibrinolysis and the reduction of thrombin formation (Blauhut et al. [1991], Dietrich et al. [1995]). Thus, inhibition of both the plasma and plasma kallikrein is important for the hemostatic effect of aprotinin.
  • Kunitz domains within the meaning of this invention are homologs of the aprotinin having 55 to 62 amino acid residues, which usually contain six cysteine residues and three disulfide bridges, each between the positions Cys 5 - Cys 55; Cys 14 - Cys 38 and Cys 30 - Cys 51 (aprotinin numbering) are formed.
  • the amino acid residues are numbered according to the 58 amino acid residues of aprotinin as shown in Table 1.
  • Some Kunitz domains contain insertions or deletions in addition to the amino acid residues shown in Table 1.
  • Kunitz inhibitors have hitherto been found, inter alia, in various vertebrates (eg human, bovine) and in some invertebrates (nudibranch, sea anemone) (Laskowski and Kato [1980] and references therein). Such naturally occurring Kunitz domains are referred to in this application as "natural Kunitz domains.” Examples of natural Kunitz domains include aprotinin, human placental bikunin domain 1, human placental Bikunin domain 2 and human TFPI-I domain 1. The sequences of some natural Kunitz domains are listed in Table 2.
  • Non-natural Kunitz domains Various unnatural Kunitz domains have been described in the literature (Dennis et al., 1995, Markland et al., 1996a). , Markland et al [1996b], Apeler et al. [2004], EP 0307592) The sequences of some non-natural Kunitz domains are shown in Table 3.
  • Aprotinin is a potent inhibitor of plasmin and plasma kallikrein (Table 4), its clinical effect is attributed to the inhibition of intrinsic blood coagulation (contact activation), the inhibition of fibrinolysis and the reduction of thrombin formation (Blauhut et al., 1991, Dietrich et al., 1995) Formation of antibodies Repeated Trasylol may cause allergic reactions (anaphylactic shock), which may increase the possibility of multiple use of aprotinin restricted (Dietrich et al. [2001], Beierlein et al. [2005]).
  • the aim of the present invention is the production of aprotinin variants with an aprotinin comparable or improved efficacy (in terms of enzyme inhibition of plasmin and plasma kallikrein, inhibition of fibrinolysis, inhibition of coagulation and reduction of bleeding time) with less antigenicity and immunogenicity.
  • aprotinin surprisingly revealed positions of amino acid residues of aprotinin whose targeted replacement with corresponding amino acid residues of other natural or non-natural Kunitz domains yield aprotinin variants which show an immunologically improved profile. These amino acid residues serve as a starting point for targeted mutagenesis (formation of chimeras, see below).
  • amino acid residues of aprotinin Tyr-10, Arg-17, He-19, Ala-40, Lys-41 and Ala-48; Also important in this analysis are the amino acid residues of aprotinin Pro-9, Lys-26, Ala-27, Leu-29, Gln-31, Arg-39, Lys-46 and Asp-50 Considered as immunologically relevant.
  • Variants of aprotinin according to the invention are formed by formation of chimeras between aprotinin and other natural or non-natural Kunitz domains.
  • the formation of chimeras occurs by replacement of one or more amino acid residues that are considered immunologically relevant.
  • amino acid residues in the aprotinin active site become more resistant to the amino acid residues of other natural or non-natural Kunitz domains exchanged to improve the effectiveness.
  • Variants of aprotinin according to the invention have the general formula: X 1 X 2 DFCLEPPX 1 OTGPCX 15 X 16 XI 7 XI 8 XI 9 RYFYNAKAGX 29 CQTFVYGGCRX 4 OX 4 IRNNFKSX 48 EDCMRTCGGA
  • X n means an amino acid residue of a Kunitz domain according to the numbering shown in Table 1. This amino acid residue can be either aprotinin or another natural or non-natural Kunitz domain listed in Tables 2 and 3. Variants of the invention are produced by exchanging at least one amino acid residue of aprotinin for another amino acid residue of the corresponding natural or unnatural Kunitz domain.
  • Preferred variants of aprotinin according to the invention inhibit plasmin with an IC50 ⁇ 30 nM.
  • Plasma kallikrein is inhibited by preferred erf ⁇ ndungssiee variants with an IC50 ⁇ 30 nM (see Table 4).
  • Preferred variants of aprotinin according to the invention show a lower cross-reactivity to sera of aprotinin-treated patients (reduced antigenicity in a cross-reactivity score 1, see Table 4) and have a reduced immunogenicity in the T-cell epitope profiling (reduced T H epitope number , see Table 5).
  • Preferred variants of aprotinin according to the invention exhibit, upon inhibition of fibrinolysis (FIGS.
  • aprotinin BPTI-mut2 (Seq No. 3), BPTI-mut5 (Seq No. 6), BPTI-mut10 (Seq No. 11) and BPTI-mutl3 (Seq No. 14) shown in FIG ). Further variants of aprotinin according to the invention are shown in FIG.
  • This invention also relates to pharmaceutical compositions containing one or more of the variants of aprotinin according to the invention.
  • the described novel aprotinin variants are suitable for the treatment of the following disease states: loss of blood in operations with an 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 (cerebral edema, spinal cord 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 skin cancer symptoms (actinic keratosis, basal cell carcinoma, squamous cell carcinoma, malignant melanoma
  • Oligonucleotides for site directed mutagenesis experiments primers for PCR (polymerase chain reaction) and sequencing reactions were purchased from the company Operon, synthetic genes (optimized for S. cerevisiae coden-usage) from Geneart.
  • In vitro mutagenesis was performed using the Quick-change II XL site-directed mutagenesis kit according to the manufacturer (Stratagene).
  • kits from Qiagen Hot Star Mastermix
  • Stratagene PfuUltra Hotstart DNA Polymerase
  • Novagen KOD HiFi, Hot Start and XL DNA Polymerases
  • Qiagen was used PCR Purification Kit from Qiagen. 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).
  • Variants of BPTI bovine pancreatic trypsin inhibitor, aprotinin
  • BPTI bovine pancreatic trypsin inhibitor, aprotinin
  • BPTI-mutl 3 generated using the Stratagene PCR kit.
  • the mutagenesis primer A (down) and primer B (up) were used.
  • the mutagenesis primer A had the following sequence: mutagenesis primer A:
  • This primer generates the mutations RIA, P2E in BPTI-mut10 (coded by GCT and GAA, in bold) and can also be used to generate mutations R1A, P2E in BPTI-mutl3, the restriction site for BsaBI (GATnnnATC) at 5 '- End of the primer is underlined.
  • Primer B :
  • the PCR mixture contained approximately 100 ng of plasmid DNA (BPTI-mut2 or BPTI-mut5), 1 OpMoI mutagenesis primer A, 1 OpMoI primer B, ImM dNTPs, IxPCR reaction buffer (Stratagene), 2.5 U PfuUltra hotstart DNA polymerase (Stratagene) in a total volume of 50 ⁇ l.
  • the 'cycle' conditions were 3 min. at 94 ° C, 30 cycles of 1 min each. at 94 ° C, 1 min. at 50 ° C., 1 min. at 72 ° C and a subsequent incubation for 5 min. at 72 ° C.
  • the PCR mixture was purified with a purification kit (Qiagen), cut with the restriction enzymes BsaBI and Sphl and ligated into the yeast secretion vector pIU10.10W, also cut with BsaBI and Sphl. E.coli DH5 ⁇ cells were transformed with the ligation mixture. From the resulting clones, the successful mutagenesis was verified by DNA sequence analysis and the variants BPTI mutlO and BPTI mutl3 used for further work.
  • Yeast cells eg strain JC34.4D (MATD, ura3-52, suc2) were grown in 10 ml YEPD (2% glucose, 2% peptone, 1% Difco yeast extract) and harvested at an ODgQO of 0.6 to 0.8 , The cells were washed with 5 ml of solution A (1 M sorbitol, 3% ethylene glycol; 10 mM bicine pH 8.35), resuspended in 0.2 ml of solution A and stored at -7O 0 C.
  • solution A (1 M sorbitol, 3% ethylene glycol; 10 mM bicine pH 8.35
  • Plasmid DNA (5 ⁇ g) and carrier DNA (50 ⁇ g of herring sperm DNA) were added to the frozen cells. The cells were then thawed by shaking for 5 min at 37 ° C. After addition of 1, 5 ml of solution B (40% PEG 1000; 200 mM bicine pH 8.35) the cells were incubated for 60 min at 30 0 C, (after pelleting with 1.5 ml of solution C 0.15 M NaCl, 10 mM bicine pH 8.35) and resuspended in 100 ⁇ l of solution C. The plating took place on a selection medium with 2% agar. Were transformants after incubation for 3 days at 3O 0 C obtained Example 3
  • the contents of the SL4 solution were dissolved in demineralised water and the pH was adjusted to 3-4 with NaOH.
  • the nutrient solution was made up to 1000 ml with demineralized water and stored in aliquots at -20 ° C.
  • the starting materials of the nutrient solutions SD2 and SC5 were prepared in demineralized water and the pH was adjusted to pH 5.5. Sterilization was carried out at 121 ° C. for 20 min. Glucose was dissolved in 1/5 of the required volume in demineralized water, sterilized separately and, after cooling, added to the remaining nutrient solution. strain stocks
  • yeast transformants were created by mixing 1 ml aliquots of a preculture with 1 ml of 80% glycerol solution and stored at -14O 0 C. Precultivations The preculture fermentations were carried out in 50 ml (for main cultures in small volume) or 1 liter shake flasks (for main cultures in medium volume) filled with 10 or 100 ml SD2 nutrient solution. The inoculation took place with a trunk preserve or with a single colony from an SD2 agar plate. The cultures were incubated with constant shaking (240 rpm) for 2 - 3 days at 28 - 30 0 C. Main culture fermentations
  • the main culture fermentations on a small scale were carried out using 1 liter shake flasks filled with 100 ml SC5 nutrient solution.
  • the inoculation was usually carried out with 3 ml of the preculture described above.
  • the cultures with continuous shaking (240 rev / min) for 4 days at 28 were - incubated 30 0 C.
  • the bioreactor system of Wave Biotech (Tageiswangen, CH) was used.
  • BPTI-mutl3 Purification of BPTI-mutl3 from the supernatants of fermented yeast cells
  • the cell-free supernatants containing BPTI-mutl3 prepared in the main culture fermentation were mixed with IM 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 at 1 ml / min to a 10 ml trypsin agarose column (Sigma-T1763).
  • BPTI-mutl 3 was eluted with 180 ml of 50 mM KCl / 10 mM HCl pH 2.0. The 2 ml fractions were collected in Collected tubes each containing 500 ul 200 raM Tris pH 7.6, 2 M NaCl to neutralize. Fractions containing BPTI-mutl3 were identified by the trypsin inhibition assay described below.
  • Trypsin-inhibiting fractions were pooled and dialysed twice in a dialysis tubing with a 1,000,000 dalton cut-off size (Spectra / POR6) against each 2 liters of 50 mM Tris pH 7.5.
  • the dialyzate was concentrated in an Amicon 8200 stirred cell over an ultrafiltration membrane of exclusion size 1, 000 daltons.
  • the protein concentration was determined with a Coomassie Plus test (Pierce, 23236) according to the manufacturer's instructions. The measured protein concentration was typically between 0.1 and 6 mg / ml.
  • the trypsin-inhibiting fractions were pooled after purification over trypsin agarose, mixed with the same volume of 0.1% TFA and applied to a Source 15 RPC column.
  • the column was washed with 6 ml of 0.1% TFA (buffer HPLC-A) and then BPTI-mutl 3 with a 25 ml gradient on 50% buffer HPLC-B (0.1% TFA, 60% acetonitrile) and another 5 ml Gradients to 100% buffer HPLC-B eluted.
  • the eluates containing BPTI-mutl3 were lyophilized and the lyophilizate was taken up in 250 ⁇ l of 50 mM Tris pH 7.5 per fraction.
  • the purified protein was diluted to 2 pmol / ⁇ l with a 0.1% TFA solution and acidified at the same time. After separation, this sample was analyzed by mass spectrometry using a GromSil 120 ODS-4 HE (3 ⁇ m, 250 ⁇ 0.2 mm). The molecular weights of BPTI mut 13 (6379.1 daltons) were unambiguously demonstrated with a delta of 0.7 daltons on the basis of the multiply charged substance ions obtained.
  • the protein after denaturation with guanidinium hydrochloride, reduction with dithiothreitol and derivatization with iodoacetamide, was cleaved by tryptic cleavage.
  • the resulting cleavage peptides were also analyzed by mass spectrometry and, based on the proven peptide masses and MS / MS spectra, accurate sequence coverage of the protein was established.
  • the inhibitory potency of BPTI-mutl3 against the enzymatic activities of trypsin, plasmin and plasma kallikrein were determined in biochemical assays in white 384-well microtiter plates using fluorogenic substrates.
  • the assay buffer was composed of 50 mM Tris / Cl, pH 7.4, 100 mM NaCl, 5 mM CaCl 2 , 0.08% (w / v) BSA.
  • the test conditions were as follows:
  • BPTI-mutl3 Per 10 ul of a serial dilution of BPTI-mutl3 were placed and pre-incubated with 20 ul 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.
  • trasylol aprotinin
  • Human citrated plasma was supplemented with 0.13 pM tissue factor (TF) and 164 U / ml tissue plasminogen activator (tPA) as well as BPTI mutl3 or
  • BPTI-mutl3 Inhibition of Coagulation by BPTI-mutl3
  • trasylol aprotinin
  • Human citrated plasma was spiked with 12 mM CaCl 2 to induce coagulation and with BPTI-mutl3 or aprotinin in various concentrations (0.1 ⁇ M to 10 ⁇ M).
  • physiological saline was used in place of the Kunitz domains.
  • 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.
  • a competitive ELISA was established.
  • a commercially available ELISA for the detection of anti-aprotinin antibodies in human serum (CellTrend, Luckenwalde) was modified so that the competition of the variants and aprotinin for binding to anti-aprotinin antibodies could be detected with high sensitivity.
  • the anti-aprotinin antibody-containing antisera were from aprotinin-treated patients who had developed a high antibody titre to the protein.
  • the antisera in the dilution buffer together with aprotinin or the BPTI variant to be tested were preincubated at room temperature for 1 h with gentle shaking in a total volume of 300 ⁇ l each. This usually came depending on the used Antiserum dilutions of 1: 3000 to 1: 7500 are used.
  • Each protein to be tested was tested at three different concentrations (1, 10, 100 nM). An empty control without aprotinin or BPTI variant was felt as a reference. After preincubation, the batch was transferred to a well precoated with aprotinin well of the microtiter modules contained in the test kit.
  • each well was washed according to the manufacturer's instructions and then a color reaction was generated with the aid of the peroxidase-coupled secondary antibody supplied.
  • the optical density was measured in a Tecan Reader at 450 nm (reference wavelength 620 nm).
  • the measured values for each protein concentration tested were set to the reference blank value (100%) in the percentage ratio.
  • Variants with cross-reactivities reduced relative to aprotinin to the antisera used were typically> 20% residual ELISA at 100 nM protein (cross-reactivity score: 1).
  • aprotinin and variants with similar or increased cross-reactivity were characterized by residual signals of ⁇ 20% at 100 nM protein (cross-reactivity score: 0).
  • HLA class II epitopes also referred to as T H epitopes.
  • the Epibase® platform analyzes all possible 10 amino acid residue long peptide portions of a target sequence to be tested for binding to 48 HLA class II receptors (allotypes are 20x DRB1, 7x DRB3 / 4/5, 14x DQ and 7x DP).
  • the free binding energy is calculated and a dissociation constant (K d ) is determined.
  • the respective peptides are classified as strong (S), middle (M) and weak or non (N) binders. The following limits were used: S: strong binders, K d ⁇ 0.1 ⁇ M; M: middle binders, 0.1 ⁇ M ⁇ IQ ⁇ 0.8 ⁇ M; N: weak or not binder, 0.8 ⁇ M ⁇ K d .
  • the observed T H cell activation / proliferation is generally interpreted as DRBI specific.
  • participation of the DRB3 / 4/5, DQ and DP can not be ruled out. Due to the lower expression rate of these genes compared to DRBl, only strong binders for DRB3 / 4/5, DQ and DP are included in the consideration of the critical epitopes. The critical epitopes are therefore strong binders compared to the DRBl, DRB3 / 4/5, DQ and DP and additionally middle binders against DRBl counted.
  • hCAVSMC Human coronary arterial vascular smooth muscle cells
  • TEBU vascular smooth muscle cells
  • M 231 medium growth medium
  • TEBU vascular smooth muscle cells
  • the plates are previously coated with vitronectin (50 ng / cm 2) (Gibco / Invitrogen, Düsseldorf, Germany). After the incubation period, one half of the confluent cell monolayer is removed. In the cell-free area of the well about 50% of the vitronectin coating is retained.
  • the growth medium is replaced by the test medium MCDB-131 / 0.2% BSA (Molecular Cellular Developmental Biology (MCDB); Basal Medium (BSA)) (Gibco / Invitrogen, Düsseldorf, Germany) and the cells are replaced with 1OnM PDGF-BB (( R & D Systems, Wiesbaden-Nordenstadt, Germany).
  • BSA Molecular Cellular Developmental Biology
  • BSA Basal Medium
  • test substances are then added in the indicated concentrations. After 24 and 48 hours of incubation, the migration distance of the cells into the free corrugation area is determined microscopically. Each measurement point represents an average of four measured regions and at least three independent experiments were performed.
  • Neutrophils are isolated from blood by standard methods.
  • the chemotaxis of neutrophils is performed in a two chamber system.
  • a HUVEC monolayer is cultured for 24 h.
  • 1x105 Neutrophils in RPMI 1640 medium, previously loaded with a fluorescent dye, are placed in the upper chamber.
  • the lower chamber contains varying concentrations of stimulus or constant stimulus concentration (IL-8: 5nM or C5a: 1OnM) and varying concentrations of the test substance.
  • the substances to be investigated are in both chambers.
  • the assay is incubated for 45 min at 37 ° C and 5% CO2. After incubation, the cells which have migrated to the lower chamber are determined (fluorescence measurement, counting).
  • Amino acid residues 1, 2, 3, 4 may be missing 15 amino acid residues 56, 57, 58 may be missing
  • cross reactivity score 0 ⁇ 20% residual signal in ELISA at 100 nM protein
  • Cross-reactivity score 1 > 20% residual signal in ELISA at 100 nM protein
  • T H epitope number per HLA gene for aprotinin and aprotinin variants Peptides which bind to several HLAs of the same gene (DRB1, DRB3 / 4/5, DQ and DP) are counted once (for definitions, see embodiment)

Abstract

The aim of the invention is the production of aprotinin variants with a comparable or improved efficacy with relation to aprotinin with reduced antigenicity and immunogenicity. The desired properties are achieved by means of the exchange of amino acid esters in the active centre and outside the active centre for the corresponding amino acid groups of other natural or unnatural Kunitz domains. A detailed analysis of the aprotinin gave surprising positions for the amino acid esters of the aprotinin, the targeted exchange of which for the corresponding amino acid groups of other natural or unnatural Kunitz domain aprotinin variants gives (Fig 1: Seq Nr. 2 to Seq Nr. 6 and Seq Nr. 10 to Seq Nr. 14), having a reduced cross-reactivity with serum aprotinin treated patients (reduced antigenicity) (Table 4), a reduced immunogenicity in T-cell epitope profiling (Fig 1: Seq Nr.10 and Seq Nr. 13) (Table 5) and a comparative or improved action with regard to enzyme inhibition of plasmin and plasma kallikrein (Table 4), inhibition of fibrinolysis (Fig. 3 and Fig. 4), inhibition of coagulation (Fig. 5 and Fig. 6), inhibition of complex inflammatory processes (Fig. 7 and Fig. 8) and a reduction of bleeding time.

Description

Aprotinin- Varianten mit verbesserten Eigenschaften Aprotinin variants with improved properties
Die vorliegende Erfindung betrifft Aprotinin-Varianten mit verbesserten immunologischen und enzyminhibitorischen Eigenschaften sowie deren Herstellung und Verwendung.The present invention relates to aprotinin variants with improved immunological and enzyme-inhibiting properties and their preparation and use.
Kunitz-Domänen sind Polypeptide, die eine Vielzahl von Serin-Proteasen mit unterschiedlicher Wirkstärke hemmen. Sie enthalten meistens drei Disulfidbrücken, die das Protein stabilisieren und seine dreidimensionale Struktur bestimmen. Die Interaktion mit der jeweiligen Serin-Protease erfolgt hauptsächlich über einen ca. 9 Aminosäurereste langen Loop im N-terminalen Bereich derKunitz domains are polypeptides 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 amino acid residues long loop in the N-terminal region of the
Kunitz-Domäne. Dieser Loop bindet an das katalytische Zentrum der Protease und verhindert so die Spaltung der entsprechenden Protease-Substrate (Laskowski und Kato [1980]; Bode und Huber [1992]).Kunitz-domain. This loop binds to the catalytic center of the protease and thus prevents cleavage of the corresponding protease substrates (Laskowski and Kato [1980], Bode and Huber [1992]).
Aprotinin, das auch als boviner pankreatischer Trypsin-Inhibitor (BPTI) bezeichnet wird (BPTI; Fig. 1 : Seq Nr. 1) gilt als Prototyp der Kunitz-Domänen (Fritz und Wunderer [1983]). Es handelt sich hierbei um ein basisches Protein von 58 Aminosäurenreste Länge, das aus verschiedenen Organen des Rindes isoliert werden kann (u.a. Pankreas, Lunge, Leber und Herz). Aprotinin wird durch drei Disulfidbrücken stabilisiert (Cys 5 - Cys 55; Cys 14 - Cys 38; Cys 30 - Cys 51) und ist u.a. ein potenter Inhibitor von Trypsin, Plasmin und Plasmakallikrein.Aprotinin, also referred to as bovine pancreatic trypsin inhibitor (BPTI) (BPTI; Figure 1: SEQ ID NO: 1), is considered a prototype of the Kunitz domains (Fritz and Wunderer [1983]). It is a basic protein of 58 amino acid residues in length that can be isolated from various bovine organs (including pancreas, lung, liver and heart). Aprotinin is stabilized by three disulfide bridges (Cys 5 - Cys 55, Cys 14 - Cys 38, Cys 30 - Cys 51) and is among others. a potent inhibitor of trypsin, plasmin and plasma kallikrein.
Durch Röntgenstrukturanalyse des Komplexes zwischen Aprotinin und Rindertrypsin konnte gezeigt werden, daß der Kontaktbereich des Aprotinins zum katalytischen Zentrum der Protease im Wesentlichen durch einen aus den Aminosäureresten 11 bis 19, bestehenden Loop gebildet wird (siehe Bode und Huber [1992] und darin aufgeführte Referenzen). Von zentraler Bedeutung für die inhibitorische Wirkung des Aprotinins ist der Aminosäurerest Lys 15, der in besonders engem Kontakt zu dem katalytisch aktiven Serinrest der Protease steht. Der Aminosäurerest Lys 15 wird daher per Definition als Pl -Rest bezeichnet (Schechter und Berger [1967]. N-terminal von Lys- 15 befinden sich die Reste P2, P3 etc., während die Aminosäurereste C-terminal von Lys-15 als Pl ', P2' etc. bezeichnet werden. Es wurde bereits früher gezeigt, daß die inhibitorische Wirkung des Aprotinins durch gezielten Austausch von Aminosäureresten im Bereich von Rest 1 1 bis Rest 19 verändert werden kann (Otlewski et al. [2001], Apeler et al. [2004], Krowarsch et al. [2005]). Für die Wirksamkeit des Aprotinins sind außerdem die Aminosäurereste 36 - 39 von Bedeutung (Fritz und Wunder [1983], Krowarsch et al. [2005]). Aprotinin wird unter dem Handelsnamen Trasylol heute hauptsächlich in der Herzchirurgie eingesetzt, nachdem klinische Studien gezeigt haben, daß eine Behandlung mit Aprotinin den Transfusionsbedarf bei derartigen Operationen signifikant verringert und zur Reduktion von Nachblutungen führt (Royston [1992]). Seine klinische Wirkung wird auf die Inhibition der intrinsischen Blutgerinnung (Kontaktaktivierung), die Hemmung der Fibrinolyse und die Reduktion der Thrombinbildung zurückgeführt (Blauhut et al. [1991], Dietrich et al. [1995]). Somit ist für die blutungsstillende Wirkung des Aprotinins die Hemmung sowohl des Plasmins als auch des Plasmakallikreins von Bedeutung.X-ray diffraction analysis of the aprotinin-bovine trypsin complex revealed that the aprotinin contact region to the protease catalytic center is essentially formed by a loop consisting of amino acid residues 11 to 19 (see Bode and Huber [1992] and references therein). , Of central importance for the inhibitory action of aprotinin is the amino acid residue Lys 15, which is in particularly close contact with the catalytically active serine residue of the protease. The amino acid residue Lys 15 is therefore designated by definition as Pl residue (Schechter and Berger [1967].) N-terminal of Lys-15 are the residues P2, P3, etc., while the amino acid residues are C-terminal of Lys-15 as Pl ', 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].) The amino acid residues 36-39 are also important for the effectiveness of aprotinin (Fritz and Wunder [1983], Krowarsch et al. [2005]). Aprotinin is sold under the trade name Trasylol is currently used mainly in cardiac surgery, after clinical studies have shown that treatment with aprotinin significantly reduces the need for transfusion in such operations and reduces rebleeding (Royston [1992]) It is attributed to the inhibition of intrinsic blood coagulation (contact activation), the inhibition of fibrinolysis and the reduction of thrombin formation (Blauhut et al. [1991], Dietrich et al. [1995]). Thus, inhibition of both the plasma and plasma kallikrein is important for the hemostatic effect of aprotinin.
Trauma, Reperfusion und extrakorporale Zirkulation sind Ursachen für komplexe inflammatorische Prozesse, wobei es zur Aktivierung humoraler und zellulärer Kaskadensysteme kommt. Diese führen zur Aktivierung von u.a. Leukozyten und Plättchen, die die klinisch beobachtbaren Nebenwirkungen, wie Ödembildung und Organschädigungen verursachen (Hess [2005]). Verschiedene Studien zeigen mögliche Effekte von Aprotinin auf den inflammatorischen Status von Bypass-Patienten (Asimakopoulos [2000]). Als bovines Protein führt Aprotinin im Menschen zur Bildung von Antikörpern. Bei wiederholter Gabe von Trasylol kann es zu allergischen Reaktionen (anaphylaktischer Schock) kommen. Das Risiko hierfür liegt bei 2.8%, wodurch die Möglichkeit einer mehrfachen Anwendung von Aprotinin eingeschränkt ist (Dietrich et al. [2001], Beierlein et al. [2005]). Es besteht somit ein hoher medizinischer Bedarf an Wirkstoffen mit einer ähnlichen oder besseren klinischen Wirkung als Aprotinin, die keine allergische Reaktion hervorrufen. Aprotinin-Varianten mit veränderten Hemmeigenschaften durch Austausch verschiedener Aminosäurereste wurden in WO 89/01968, WO 89/10374, EP 0 307 592, EP 683 229 und DE-PS 3 339 693 beschrieben. Aus Gründen der besseren technischen Herstellbarkeit ist es günstig, in bestimmten Fällen, eine Modifikation am N-terminalen Ende des Inhibitormoleküls vorzunehmen. Solche Modifikationen können N-terminale Verkürzungen oder Verlängerungen oder Deletionen von einer oder mehrerer Aminosäurereste sein. N-terminal modifizierte Aprotinin-Varianten wurden in EP 419 878 beschrieben.Trauma, reperfusion and extracorporeal circulation are the causes of complex inflammatory processes, with activation of humoral and cellular cascade systems. These lead to the activation of u.a. Leukocytes and platelets causing the clinically observable side effects such as edema and organ damage (Hess [2005]). Various studies show possible effects of aprotinin on the inflammatory status of bypass patients (Asimakopoulos [2000]). 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. Aprotinin variants with altered inhibitory properties by exchanging various amino acid residues have been described in WO 89/01968, WO 89/10374, EP 0 307 592, EP 683 229 and DE-PS 3 339 693. For reasons of better technical manufacturability, it is advantageous, in certain cases, to carry out a modification at the N-terminal end of the inhibitor molecule. Such modifications may be N-terminal truncations or extensions or deletions of one or more amino acid residues. N-terminally modified aprotinin variants have been described in EP 419 878.
Detaillierte Beschreibung der ErfindungDetailed description of the invention
Kunitz-Domänen im Sinne dieser Erfindung sind Homologe des Aprotinins mit 55 bis 62 Aminosäureresten, die meistens sechs Cysteinreste und drei Disulfidbrücken enthalten, die jeweils zwischen den Positionen Cys 5 - Cys 55; Cys 14 - Cys 38 sowie Cys 30 - Cys 51 (Aprotinin Numerierung) ausgebildet sind. Die Aminosäurereste werden, wie in Tabelle 1 dargestellt, entsprechend den 58 Aminosäureresten des Aprotinins numeriert. Einige Kunitz-Domänen enthalten zusätzlich zu den in Tabelle 1 angegebenen Aminosäureresten Insertionen oder Deletionen.Kunitz domains within the meaning of this invention are homologs of the aprotinin having 55 to 62 amino acid residues, which usually contain six cysteine residues and three disulfide bridges, each between the positions Cys 5 - Cys 55; Cys 14 - Cys 38 and Cys 30 - Cys 51 (aprotinin numbering) are formed. The amino acid residues are numbered according to the 58 amino acid residues of aprotinin as shown in Table 1. Some Kunitz domains contain insertions or deletions in addition to the amino acid residues shown in Table 1.
Kunitz-Inhibitoren wurden bisher u.a. in verschiedenen Vertebraten (z.B. Mensch, Rind) sowie in einigen Invertebraten (Nacktschnecke, Seeanemone) gefunden (Laskowski und Kato [1980] und darin aufgeführte Referenzen). Solche in der Natur vorkommenden Kunitz-Domänen werden in dieser Anmeldung als „natürliche Kunitz-Domänen" bezeichnet. Beispiele für natürliche Kunitz- Domänen sind u.a. Aprotinin, humanes placentales Bikunin Domäne 1 , humanes placentales Bikunin Domäne 2 und humanes TFPI-I Domäne 1. Die Sequenzen einiger natürlicher Kunitz- Domänen sind in Tabelle 2 aufgeführt.Kunitz inhibitors have hitherto been found, inter alia, in various vertebrates (eg human, bovine) and in some invertebrates (nudibranch, sea anemone) (Laskowski and Kato [1980] and references therein). Such naturally occurring Kunitz domains are referred to in this application as "natural Kunitz domains." Examples of natural Kunitz domains include aprotinin, human placental bikunin domain 1, human placental Bikunin domain 2 and human TFPI-I domain 1. The sequences of some natural Kunitz domains are listed in Table 2.
Mit Hilfe gentechnischer Methoden ist es möglich, rekombinante Varianten von Kunitz-Domänen herzustellen, die sich von natürlichen Kunitz-Domänen in einer oder in mehreren Aminosäuren unterscheiden. Solche durch Austausch von Aminosäuren entstandenen Kunitz-Domänen werden in dieser Anmeldung als „nicht-natürliche Kunitz-Domänen" bezeichnet. Verschiedene nichtnatürliche Kunitz-Domänen wurden in der Literatur beschrieben (Dennis et al. [1995], Markland et al. [1996a], Markland et al. [1996b], Apeler et al. [2004], EP 0307592). In Tabelle 3 sind die Sequenzen einiger nicht-natürlicher Kunitz-Domänen dargestellt. Aprotinin ist ein potenter Inhibitor von Plasmin und Plasmakallikrein (Tabelle 4), dessen klinische Wirkung auf die Inhibition der intrinsischen Blutgerinnung (Kontaktaktivierung), die Hemmung der Fibrinolyse und die Reduktion der Thrombinbildung zurückgeführt wird (Blauhut et al. [1991], Dietrich et al. [1995]). Als bovines Protein führt Aprotinin im Menschen zur Bildung von Antikörpern. Bei wiederholter Gabe von Trasylol kann es zu allergischen Reaktionen (anaphylaktischer Schock) kommen, wodurch die Möglichkeit einer mehrfachen Anwendung von Aprotinin eingeschränkt ist (Dietrich et al. [2001], Beierlein et al. [2005]).Using genetic engineering methods, it is possible to produce recombinant variants of Kunitz domains that differ from natural Kunitz domains in one or more amino acids. Such Kunitz domains resulting from the exchange of amino acids are referred to in this application as "non-natural Kunitz domains." Various unnatural Kunitz domains have been described in the literature (Dennis et al., 1995, Markland et al., 1996a). , Markland et al [1996b], Apeler et al. [2004], EP 0307592) The sequences of some non-natural Kunitz domains are shown in Table 3. Aprotinin is a potent inhibitor of plasmin and plasma kallikrein (Table 4), its clinical effect is attributed to the inhibition of intrinsic blood coagulation (contact activation), the inhibition of fibrinolysis and the reduction of thrombin formation (Blauhut et al., 1991, Dietrich et al., 1995) Formation of antibodies Repeated Trasylol may cause allergic reactions (anaphylactic shock), which may increase the possibility of multiple use of aprotinin restricted (Dietrich et al. [2001], Beierlein et al. [2005]).
Ziel der vorliegenden Erfindung ist die Herstellung von Aprotinin-Varianten mit einer dem Aprotinin vergleichbaren oder verbesserten Wirksamkeit (in Bezug auf Enzyminhibition von Plasmin und Plasmakallikrein, Inhibition der Fibrinolyse, Inhibition der Koagulation und Verringerung der Blutungszeit) bei geringerer Antigenizität und Immunogenizität. Eine detaillierte Analyse des Aprotinins ergab überraschenderweise Positionen von Aminosäureresten des Aprotinins, deren gezielter Austausch gegen entsprechende Aminosäurereste anderer natürlicher oder nicht-natürlicher Kunitz-Domänen Aprotinin-Varianten ergibt, die ein immunologisch verbessertes Profil zeigen. Diese Aminosäurereste dienen als Ausgangspunkt der gezielten Mutagenese (Bildung von Chimären, siehe unten). Besonders hervorzuheben sind bei dieser Analyse die Aminosäurereste des Aprotinins: Tyr-10, Arg-17, He- 19, Ala-40, Lys-41 und Ala-48; ebenfalls von Bedeutung sind bei dieser Analyse die Aminosäurereste des Aprotinins Pro-9, Lys-26, Ala-27, Leu-29, Gln-31 , Arg-39, Lys-46 und Asp- 50. Die gefundenen Aminosäurereste werden im Rahmen dieser Erfindung als immunologisch relevant betrachtet.The aim of the present invention is the production of aprotinin variants with an aprotinin comparable or improved efficacy (in terms of enzyme inhibition of plasmin and plasma kallikrein, inhibition of fibrinolysis, inhibition of coagulation and reduction of bleeding time) with less antigenicity and immunogenicity. A detailed analysis of aprotinin surprisingly revealed positions of amino acid residues of aprotinin whose targeted replacement with corresponding amino acid residues of other natural or non-natural Kunitz domains yield aprotinin variants which show an immunologically improved profile. These amino acid residues serve as a starting point for targeted mutagenesis (formation of chimeras, see below). Particularly noteworthy in this analysis are the amino acid residues of aprotinin: Tyr-10, Arg-17, He-19, Ala-40, Lys-41 and Ala-48; Also important in this analysis are the amino acid residues of aprotinin Pro-9, Lys-26, Ala-27, Leu-29, Gln-31, Arg-39, Lys-46 and Asp-50 Considered as immunologically relevant.
Erfϊndungsgemäße Varianten des Aprotinins entstehen durch Bildung von Chimären zwischen Aprotinin und anderen natürlichen oder nicht-natürlichen Kunitz-Domänen. Die Bildung von Chimären erfolgt durch Austausch einer oder mehrerer Aminosäurereste, die als immunologisch relevant betrachtet werden. In einigen Fällen werden zusätzlich Aminosäurereste im aktiven Zentrum des Aprotinins gegen die Aminosäurereste anderer natürlicher oder nicht-natürlicher Kunitz-Domänen ausgetauscht, um die Wirksamkeit zu verbessern. Aus Gründen der besseren technischen Herstellbarkeit ist es außerdem günstig, in bestimmten Fällen, eine Modifikation am N-terminalen Ende des Inhibitormoleküls vorzunehmen So führen z.B. die Austausche RIA und P2E zu einer verbesserten und einheitlichen Prozessierung der Inhibitoren in Hefezellen ohne die gleichzeitige Generierung neuer bzw. zusätzlicher THeifer-Zell-Epitope (Fig.l : Seq Nr. 10 bis Seq Nr. 14). Überraschenderweise konnten auf diese Weise Aprotinin-Varianten erzeugt werden, die eine dem Aprotinin vergleichbare oder verbesserte Wirksamkeit bei geringerer Antigenizität und Immunogenizität besitzen (Fig.l : Seq Nr. 2 bis Seq Nr. 6 und Seq Nr. 10 bis Seq Nr. 14). Erfindungsgemäße Varianten des Aprotinins haben die allgemeine Formel: X1X2DFCLEPPX1OTGPCX15X16XI7XI8XI9RYFYNAKAGX29CQTFVYGGCRX4OX4IRNNFKSX48 EDCMRTCGGAVariants of aprotinin according to the invention are formed by formation of chimeras between aprotinin and other natural or non-natural Kunitz domains. The formation of chimeras occurs by replacement of one or more amino acid residues that are considered immunologically relevant. In addition, in some cases, amino acid residues in the aprotinin active site become more resistant to the amino acid residues of other natural or non-natural Kunitz domains exchanged to improve the effectiveness. For reasons of better technical manufacturability, it is also advantageous, in certain cases, to make a modification at the N-terminal end of the inhibitor molecule. For example, the exchanges RIA and P2E lead to an improved and uniform processing of the inhibitors in yeast cells without the simultaneous generation of new or additional T He he fer cell epitopes (Fig.l: Seq no. 10 to seq no. 14). Surprisingly, it was possible in this way to produce aprotinin variants which have a comparable or improved aprotinin activity with lower antigenicity and immunogenicity (FIG. 1: Seq No. 2 to Seq No. 6 and Seq No. 10 to Seq No. 14) , Variants of aprotinin according to the invention have the general formula: X 1 X 2 DFCLEPPX 1 OTGPCX 15 X 16 XI 7 XI 8 XI 9 RYFYNAKAGX 29 CQTFVYGGCRX 4 OX 4 IRNNFKSX 48 EDCMRTCGGA
Hierbei bedeutet ,,Xn" ein Aminosäurerest einer Kunitz-Domäne nach der in Tabelle 1 dargestellten Numerierung. Dieser Aminosäurerest kann entweder Aprotinin oder einer anderen, in den Tabellen 2 und 3 aufgeführten natürlichen oder nicht-natürlichen Kunitz-Domäne entstammen. Erfindungsgemäße Varianten entstehen durch den Austausch mindestens eines Aminosäurerestes von Aprotinin gegen einen anderen Aminosäurerest der entsprechenden natürlichen oder nichtnatürlichen Kunitz-Domäne.Herein, "X n " means an amino acid residue of a Kunitz domain according to the numbering shown in Table 1. This amino acid residue can be either aprotinin or another natural or non-natural Kunitz domain listed in Tables 2 and 3. Variants of the invention are produced by exchanging at least one amino acid residue of aprotinin for another amino acid residue of the corresponding natural or unnatural Kunitz domain.
Bevorzugte erfindungsgemäße Varianten des Aprotinins hemmen Plasmin mit einem IC50 < 30 nM. Plasmakallikrein wird durch bevorzugte erfϊndungsgemäße Varianten mit einem IC50 < 30 nM gehemmt (siehe Tabelle 4). Bevorzugte erfindungsgemäße Varianten des Aprotinins zeigen gegenüber Seren Aprotinin-behandelter Patienten eine geringere Kreuzreaktivität (verringerte Antigenizität bei einem Kreuzreaktivitätsscore 1 , siehe Tabelle 4) und weisen in der T-Zell-Epitop- Profilierung eine verringerte Immunogenizität auf (verringerte TH-Epitop-Anzahl, siehe Tabelle 5). Bevorzugte erfindungsgemäße Varianten des Aprotinins zeigen bei Inhibition der Fibrinolyse (Fig. 3 und Fig. 4), Inhibition der Koagulation (Fig. 5 und Fig.6), Inhibition der IL-8 induzierten Chemotaxis von Neutrophilen (Fig. 7) und Inhibition der horizontalen Migration von hCASMC (Fig. 8) eine dem Aprotinin vergleichbare oder bessere Wirkung.Preferred variants of aprotinin according to the invention inhibit plasmin with an IC50 <30 nM. Plasma kallikrein is inhibited by preferred erfϊndungsgemäße variants with an IC50 <30 nM (see Table 4). Preferred variants of aprotinin according to the invention show a lower cross-reactivity to sera of aprotinin-treated patients (reduced antigenicity in a cross-reactivity score 1, see Table 4) and have a reduced immunogenicity in the T-cell epitope profiling (reduced T H epitope number , see Table 5). Preferred variants of aprotinin according to the invention exhibit, upon inhibition of fibrinolysis (FIGS. 3 and 4), inhibition of coagulation (FIGS. 5 and 6), inhibition of IL-8 induced chemotaxis of neutrophils (FIG. 7) and inhibition of horizontal migration of hCASMC (Figure 8) a comparable or better aprotinin effect.
Besonders bevorzugt sind die in Fig.l dargestellten Varianten von Aprotinin BPTI-mut2 (Seq Nr. 3), BPTI-mut5 (Seq Nr. 6), BPTI-mutlO (Seq Nr. 11) und BPTI-mutl3 (Seq Nr. 14). Weitere erfindungsgemäße Varianten von Aprotinin sind in Fig. 1 dargestellt.Especially preferred are the variants of aprotinin BPTI-mut2 (Seq No. 3), BPTI-mut5 (Seq No. 6), BPTI-mut10 (Seq No. 11) and BPTI-mutl3 (Seq No. 14) shown in FIG ). Further variants of aprotinin according to the invention are shown in FIG.
Diese Erfindung betrifft auch Arzneimittel, die eine oder mehrere der erfindungsgemäßen Varianten von Aprotinin enthalten.This invention also relates to pharmaceutical compositions containing one or more of the variants of aprotinin according to the invention.
Die beschriebenen neuartigen Aprotinin-Varianten eignen sich für die Behandlung von folgenden Krankheitszuständen: Blutverlust bei Operationen mit erhöhtem Blutungsrisiko; Therapie thromboembolischer Zustände (z. B. nach Operationen, Unfällen), Schock, Polytrauma, Sepsis, disseminierte intravasale Gerinnung (DIC), Multiorganversagen (MOF), instabile Angina, Herzinfarkt, Schlaganfall, Embolie, tiefe Venenthrombosen, entzündliche Erkrankungen (z.B. Rheuma, Asthma), invasives Tumorwachstum und Metastasierung, Schmerz-und Ödemtherapie (Gehirnödem, Rückenmarksödem) , Verhinderung der Aktivierung der Hämostase bei Dialysebehandlung, Behandlung von Symptomen der Hautalterung (Elastose, Atrophie, Faltenbildung, Gefäßveränderungen, Pigmentveränderungen, aktinische Keratose, Mitesser, Zysten), Wundheilung, Hautkrebs, Behandlung von Symptomen des Hautkrebses (aktinische Keratose, Basalzellkarzinom, Schuppenzellkarzinom, malignes Melanom), multiple Sklerose, Fibrose, Gehirnblutung, Entzündungen des Gehirns und des Rückenmarks, Infektionen des Gehirns. The described novel aprotinin variants are suitable for the treatment of the following disease states: loss of blood in operations with an 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 (cerebral edema, spinal cord 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 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.
BeispieleExamples
Beispiel 1example 1
Klonierung von BPTI-Varianten Routinemäßige Klonierungsarbeiten wurden nach Sambrook et al. (Molecular Cloning CoId Spring Harbor,1989) durchgeführt. Für die Isolation von Plasmid-DNA aus E. coli (sog. mini- und midipreps) wurden Qiagen-tips (Qiagen) verwendet. Als Wirtsorganismus für Transformationen wurde der E.coli Stamm DH5α (Stratagene) eingesetzt. Die Extraktion aus Agarosegelen wurde mit Hilfe des Qiagen gel extraction kits nach Angaben des Herstellers (Qiagen) durchgeführt. Oligonukleotide für 'site directed mutagenesis' Experimente, Primer für PCR - (Polymerase Chain Reaktion) und Sequenzierungsreaktionen wurden von der Firma Operon bezogen, synthetische Gene (optimiert für S.cerevisiae coden-usage) von der Firma Geneart. In-vitro Mutagenesen wurden mit Hilfe des Quick-change II XL Site-directed Mutagenesis Kits nach Angaben des Herstellers (Stratagene) durchgeführt. Für PCR-Experimente wurden Kits der Firma Qiagen (Hot Star Mastermix ), Stratagene ( PfuUltra Hotstart DNA Polymerase ) oder Novagen ( KOD HiFi, Hot Start and XL DNA Polymerases) nach Angaben der Hersteller eingesetzt, zur Aufreinigung von PCR-Fragmenten wurde der Qiagen PCR Purification Kit der Firma Qiagen verwendet. Alle Vektorkonstruktionen und Mutagenesen wurden mittels Cycle-DNA-Sequenzierung mit Fluoreszenz-markierten Terminatoren (Big Dye Terminator, Version 1.1, Firma Applied Biosystems) auf einem Sequenzer (3100 Avant Genetic Analyzer, Firma Applied Biosystem) bestätigt.Cloning of BPTI Variants Routine cloning work was performed according to Sambrook et al. (Molecular Cloning Co., Spring Harbor, 1989). 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 of the E. coli strain DH5α (Stratagene) was used. The extraction from agarose gels was carried out with the aid of the Qiagen gel extraction kit according to the manufacturer (Qiagen). Oligonucleotides for site directed mutagenesis experiments, primers for PCR (polymerase chain reaction) and sequencing reactions were purchased from the company Operon, synthetic genes (optimized for S. cerevisiae coden-usage) from Geneart. In vitro mutagenesis was performed using the Quick-change II XL site-directed mutagenesis kit according to the manufacturer (Stratagene). For PCR experiments, kits from Qiagen (Hot Star Mastermix), Stratagene (PfuUltra Hotstart DNA Polymerase) or Novagen (KOD HiFi, Hot Start and XL DNA Polymerases) were used according to the manufacturer's instructions. For the purification of PCR fragments, Qiagen was used PCR Purification Kit from Qiagen. 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).
Varianten von BPTI (bovine pancreatic trypsin inhibitor, Aprotinin) wurden als synthetische Gene - optimiert für S.cerevisiae codon-uasge - von der Firma Geneart bezogen. 5' und 3' zur kodierenden Region befinden sich weitere Sequenzen, die für die in-frame Subklonierungen in den Hefe-Sekretionsvektor pIUl 0.10.W (Apeler [2005]) benötigt werden.Variants of BPTI (bovine pancreatic trypsin inhibitor, aprotinin) were obtained as synthetic genes - optimized for S. cerevisiae codon-uasge - from Geneart. 5 'and 3' to the coding region are further sequences that are required for the in-frame subcloning into the yeast secretion vector pIUl 0.10.W (Apeler [2005]).
Kloniert wurden die Varianten jeweils in den Vektor pPCR-Script, der ebenfalls von der Firma Geneart bezogen wurde. Zur Übersicht der Aminosäuresequenzen von BPTI (Aprotinin) und dessen Varianten BPTI-mutl bis -mutlό siehe Fig. 1The variants were cloned in each case in the vector pPCR-Script, which was also obtained from the company Geneart. For a review of the amino acid sequences of BPTI (aprotinin) and its variants BPTI-mutl to -mutlό see FIG. 1
BPTI-mut2 (Seq Nr. 3)BPTI mut2 (Seq # 3)
DNA-Sequenz des synthetischen Gens für BPTI-mut2; die kodierende Region ist fett gedruckt, Erkennungssequenzen für Restriktionsenzyme (5': Kpnl GGTACC, BsaBI GATnnnnATC; 3': Xhol CTCGAG, Sacl GAGCTC) sind unterstrichen GGGCGAATTGGGTACCGATTCCCATCTATTTTTACTGCTGTTTTGTTTGCTGCTTCTT CTDNA sequence of the synthetic gene for BPTI mut2; the coding region is in bold, restriction enzyme recognition sequences (5 ': KpnI GGTACC, BsaBI GATnnnnATC, 3': Xhol CTCGAG, Sacl GAGCTC) are underlined GGGCGAATTGGGTACCGATTCCCATCTATTTTTACTGCTGTTTTGTTTGCTGCTTCTT CT
GCTTTGGCTAGACCAGATTTTTGTTTGGAACCACCAGATACTGGTCCATGTAAAG CTGCTGCTTTGGCTAGACCAGATTTTTGTTTGGAACCACCAGATACTGGTCCATGTAAAG CTGCT
ATTAAAAGATATTTCTATAATGCTAAAGCTGGTTTGTGTCAAACTTTTGTTTATGG TGGTATTAAAAGATATTTCTATAATGCTAAAGCTGGTTTGTGTCAAACTTTTGTTTATGG TGGT
TGTAGAGGTAATAGAAATAACTTTAAATCTTTGGAAGATTGTATGAGAACTTGTG GTGGT GCTTAATAACTCGAGCTCCAGCTTTTGTTCCCTGTAGAGGTAATAGAAATAACTTTAAATCTTTGGAAGATTGTATGAGAACTTGTG GTGGT GCTTAATAACTCGAGCTCCAGCTTTTGTTCCC
BPTI-mut5 (Seq Nr. 6)BPTI-mut5 (Seq No. 6)
DNA-Sequenz des synthetischen Gens für BPTI-mut5; die kodierende Region ist fett gedruckt, Erkennungssequenzen für Restriktionsenzyme ( 5': Kpnl GGTACC, BsaBI GATnnnnATC; 3': Xhol CTCGAG, Sacl GAGCTC ) sind unterstrichenDNA sequence of the synthetic gene for BPTI mut5; the coding region is in bold, restriction enzyme recognition sequences (5 ': KpnI GGTACC, BsaBI GATnnnnATC, 3': Xhol CTCGAG, Sacl GAGCTC) are underlined
GGGCGAATTGGGTACCGATTCCCATCTATTTTTACTGCTGTTTTGTTTGCTGCTTCTTGGGCGAATTGGGTACCGATTCCCATCTATTTTTACTGCTGTTTTGTTTGCTGCTTCTT
CTCT
GCTTTGGCTAGACCAGATTTTTGTTTGGAACCACCAGAAACTGGTCCATGTAGAG CTGCTGCTTTGGCTAGACCAGATTTTTGTTTGGAACCACCAGAAACTGGTCCATGTAGAG CTGCT
CATCCAAGATATTTCTATAATGCTAAAGCTGGTTCTTGTCAAACTTTTGTTTATGGCATCCAAGATATTTCTATAATGCTAAAGCTGGTTCTTGTCAAACTTTTGTTTATGG
TGGTTGGT
TGTAGAGGTAATAGAAATAACTTTAAATCTGAAGAAGATTGTATGAGAACTTGTGTGTAGAGGTAATAGAAATAACTTTAAATCTGAAGAAGATTGTATGAGAACTTGTG
GTGGT GCTTAATAACTCGAGCTCCAGCTTTTGTTCCCGTGGT GCTTAATAACTCGAGCTCCAGCTTTTGTTCCC
Subkloniert wurden die synthetischen Gene in den modifizierten Hefe-Sekretionsvektor pIUlO.lO.W (Apeler [2005[) über die Restriktionsschnittstellen BsaBI und Xhol und anschließend kompetente E.coli DH5α Zellen damit transformiert. BPTI-mutlO (Seq Nr. 11) und BPTI-mutl3 (Seq Nr. 14)The synthetic genes were subcloned into the modified yeast secretion vector pIU10.10.W (Apeler [2005 [) via the restriction sites BsaBI and Xhol and subsequently transformed to competent E. coli DH5α cells. BPTI-mutlO (Seq # 11) and BPTI-mutl3 (Seq # 14)
Ausgehend von BPTI-mut2 bzw. BPTI-mut5 wurden durch PCR die Varianten BPTI-mutlO bzw.Starting from BPTI-mut2 or BPTI-mut5 the variants BPTI-mutlO or
BPTI-mutl 3 erzeugt unter der Verwndung des PCR-kits der Firma Stratagene. Eingesetzt wurden dabei der Mutagenese-Primer A (down) und Primer B (up). Der Mutagenese-Primer A hatte folgende Sequenz: Mutagenese-Primer A:BPTI-mutl 3 generated using the Stratagene PCR kit. The mutagenesis primer A (down) and primer B (up) were used. The mutagenesis primer A had the following sequence: mutagenesis primer A:
5'-5 '
TTAGATTCCCATCTATTITTACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCTGCTG AAG ATTTTTGTTTGG AACC AC-3 ' Dieser Primer generiert die Mutationen RIA, P2E in BPTI-mutlO (kodiert durch GCT und GAA, fett gedruckt) und kann ebenfalls zur Generierung der Mutationen R1A,P2E in BPTI-mutl3 eingesetzt werden, die Restriktionsschnittstelle für BsaBI (GATnnnnATC) am 5 '-Ende desPrimers ist unterstrichen. Primer B:TTAGATTCCCATCTATTITTACTGCTGTTTTGTTTGCTGCTTCTTCTGCTTTGGCTGCTG AAG ATTTTTGTTTGG AACC AC-3 ' This primer generates the mutations RIA, P2E in BPTI-mut10 (coded by GCT and GAA, in bold) and can also be used to generate mutations R1A, P2E in BPTI-mutl3, the restriction site for BsaBI (GATnnnnATC) at 5 '- End of the primer is underlined. Primer B:
5'- CTCTAGATGCATGCTCGAGTTATTA-3'5'-CTCTAGATGCATGCTCGAGTTATTA-3 '
Die Restriktionsschnittstelle für Sphl (GCATGC) ist unterstrichen.The restriction site for Sphl (GCATGC) is underlined.
Der PCR-Ansatz enthielt ca.100 ng Plasmid-DNA (BPTI-mut2 bzw. BPTI-mut5), 1 OpMoI Mutagenese-Primer A, 1 OpMoI Primer B, ImM dNTPs, IxPCR Reaktionspuffer (Stratagene), 2.5 U PfuUltra hotstart DNA Polymerase (Stratagene) in einem Gesamtvolumen von 50μl. Die 'Cycle' -Bedingungen waren 3 min. bei 94°C, 30 Zyklen mit jeweils 1 min. bei 94°C, 1 min. bei 500C, 1 min. bei 72°C und eine anschließende Inkubation für 5 min. bei 720C. Der PCR-Ansatz wurde mit einem Purification-kit (Qiagen) aufgereinigt, mit den Restriktionsenzymen BsaBI und Sphl geschnittenund in den ebenfalls mit BsaBI und Sphl geschnittenen Hefe-Sekretionsvektor pIUlO.lO.W ligiert. Mit dem Ligationsansatz wurden E.coli DH5α Zellen transformiert. Von den so entstandenen Klonen wurde die erfolgreiche Mutagenese durch DNA-Sequenzanalyse verifiziert und die Varianten BPTI-mutlO und BPTI-mutl3 für weitere Arbeiten eingesetzt.The PCR mixture contained approximately 100 ng of plasmid DNA (BPTI-mut2 or BPTI-mut5), 1 OpMoI mutagenesis primer A, 1 OpMoI primer B, ImM dNTPs, IxPCR reaction buffer (Stratagene), 2.5 U PfuUltra hotstart DNA polymerase (Stratagene) in a total volume of 50μl. The 'cycle' conditions were 3 min. at 94 ° C, 30 cycles of 1 min each. at 94 ° C, 1 min. at 50 ° C., 1 min. at 72 ° C and a subsequent incubation for 5 min. at 72 ° C. The PCR mixture was purified with a purification kit (Qiagen), cut with the restriction enzymes BsaBI and Sphl and ligated into the yeast secretion vector pIU10.10W, also cut with BsaBI and Sphl. E.coli DH5α cells were transformed with the ligation mixture. From the resulting clones, the successful mutagenesis was verified by DNA sequence analysis and the variants BPTI mutlO and BPTI mutl3 used for further work.
Beispiel 2Example 2
Transformation von Saccbaromyces cerevisiaeTransformation of Saccbaromyces cerevisiae
Hefezellen, z.B. der Stamm JC34.4D (MATD, ura3-52, suc2) wurden in 10 ml YEPD (2 % Glucose; 2 % Pepton; 1 % Difco Hefeextrakt) angezogen und bei einer ODgQO von 0,6 bis 0,8 geerntet. Die Zellen wurden mit 5 ml Lösung A (1 M Sorbitol; 10 mM Bicin pH 8,35; 3 % Ethylenglycol) gewaschen, in 0,2 ml Lösung A resuspendiert und bei -7O0C gelagert.Yeast cells, eg strain JC34.4D (MATD, ura3-52, suc2) were grown in 10 ml YEPD (2% glucose, 2% peptone, 1% Difco yeast extract) and harvested at an ODgQO of 0.6 to 0.8 , The cells were washed with 5 ml of solution A (1 M sorbitol, 3% ethylene glycol; 10 mM bicine pH 8.35), resuspended in 0.2 ml of solution A and stored at -7O 0 C.
Plasmid DNA (5 μg) und Carrier DNA (50 μg DNA aus Herringssperma) wurden zu den gefrorenen Zellen gegeben. Die Zellen wurden anschließend durch Schütteln für 5 min bei 37°C aufgetaut. Nach Zugabe von 1 ,5 ml Lösung B (40 % PEG 1000; 200 mM Bicin pH 8,35) wurden die Zellen für 60 min bei 300C inkubiert, nach dem Pelletieren mit 1,5 ml Lösung C (0,15 M NaCl; 10 mM Bicin pH 8,35) gewaschen und in 100 μl Lösung C resuspendiert. Die Ausplattierung erfolgte auf einem Selektionsmedium mit 2 % Agar. Transformanden wurden nach einer Inkubation von 3 Tagen bei 3O0C erhalten Beispiel 3Plasmid DNA (5 μg) and carrier DNA (50 μg of herring sperm DNA) were added to the frozen cells. The cells were then thawed by shaking for 5 min at 37 ° C. After addition of 1, 5 ml of solution B (40% PEG 1000; 200 mM bicine pH 8.35) the cells were incubated for 60 min at 30 0 C, (after pelleting with 1.5 ml of solution C 0.15 M NaCl, 10 mM bicine pH 8.35) and resuspended in 100 μl of solution C. The plating took place on a selection medium with 2% agar. Were transformants after incubation for 3 days at 3O 0 C obtained Example 3
Herstellung von BPTI-mutl3 durch Fermentation der HefezellenProduction of BPTI-mutl3 by fermentation of the yeast cells
Nährlösungennutrient solutions
Zur Fermentation von Hefezellen zur Expression von BPTI-mutl3 wurden die folgenden Nährlösungen verwendet:For fermentation of yeast cells for expression of BPTI-mutl3, the following nutrient solutions were used:
Figure imgf000011_0001
Figure imgf000011_0001
Spurenelementelösung 4 (SL4-Lösung):Trace element solution 4 (SL4 solution):
Titriplex III (Merck 8418) 5 gTitriplex III (Merck 8418) 5 g
FeSO4 • 7H2O (Merck 3965) 2 gFeSO 4 • 7H 2 O (Merck 3965) 2 g
ZnSO4 • 7H2O (Merck 8883) 0.1 gZnSO 4 • 7H 2 O (Merck 8883) 0.1 g
MnCl2 • 4H2O (Merck 5927) 30 mgMnCl 2 • 4H 2 O (Merck 5927) 30 mg
H3BO3 (Merck 165) 0.3 gH 3 BO 3 (Merck 165) 0.3 g
CoCl2 • 6H2O (Merck 2533) 0.2 gCoCl 2 • 6H 2 O (Merck 2533) 0.2 g
CuCl2 • 2H2O (Merck 2733) 10 mgCuCl 2 • 2H 2 O (Merck 2733) 10 mg
NiCl2 • 6H2O (Merck 6717) 20 mgNiCl 2 • 6H 2 O (Merck 6717) 20 mg
Na2MoO4 • 2H2O (Merck 6521) 30 mgNa 2 MoO 4 • 2H 2 O (Merck 6521) 30 mg
Die Inhaltsstoffe der SL4-Lösung wurden in demineralisiertem Wasser gelöst und der pH-Wert wurde mit NaOH auf pH 3 - 4 eingestellt. Die Nährlösung wurde mit demineralisiertem Wasser auf 1000 ml aufgefüllt und in Aliquots bei -20°C gelagert.The contents of the SL4 solution were dissolved in demineralised water and the pH was adjusted to 3-4 with NaOH. The nutrient solution was made up to 1000 ml with demineralized water and stored in aliquots at -20 ° C.
Die Einsatzstoffe der Nährlösungen SD2 und SC5 wurden in demineralisiertem Wasser angesetzt und der pH-Wert auf pH 5.5 eingestellt. Die Sterilisation erfolgte für 20 min bei 121 0C. Glucose wurde in 1/5 des erforderlichen Volumens in demineralisiertem Wasser gelöst, getrennt sterilisiert und nach dem Abkühlen zur übrigen Nährlösung gegeben. StammkonservenThe starting materials of the nutrient solutions SD2 and SC5 were prepared in demineralized water and the pH was adjusted to pH 5.5. Sterilization was carried out at 121 ° C. for 20 min. Glucose was dissolved in 1/5 of the required volume in demineralized water, sterilized separately and, after cooling, added to the remaining nutrient solution. strain stocks
Stammkonserven aller Hefetransformanden wurden durch Vermischung von 1 ml-Aliquots einer Vorkultur mit 1 ml 80% Glycerin-Lösung und Lagerung bei -14O0C angelegt. Vorkulturen Die Vorkulturfermentationen wurden in 50 ml- (für Hauptkulturen im kleinen Volumen) bzw. 1 Liter- Schüttelkolben (für Hauptkulturen im mittleren Volumen), gefüllt mit 10 bzw. 100 ml SD2- Nährlösung durchgeführt. Die Beimpfung erfolgte mit einer Stammkonserve oder mit einer Einzelkolonie von einer SD2-Agarplatte. Die Kulturen wurden unter ständigem Schütteln (240 rpm) für 2 - 3 Tage bei 28 - 30 0C inkubiert. HauptkulturfermentationenStrain stocks of all the yeast transformants were created by mixing 1 ml aliquots of a preculture with 1 ml of 80% glycerol solution and stored at -14O 0 C. Precultivations The preculture fermentations were carried out in 50 ml (for main cultures in small volume) or 1 liter shake flasks (for main cultures in medium volume) filled with 10 or 100 ml SD2 nutrient solution. The inoculation took place with a trunk preserve or with a single colony from an SD2 agar plate. The cultures were incubated with constant shaking (240 rpm) for 2 - 3 days at 28 - 30 0 C. Main culture fermentations
Die Hauptkulturfermentationen im kleinen Maßstab erfolgte unter Verwendung von 1-Ltr.- Schüttelkolben gefüllt mit 100 ml SC5-Nährlösung. Die Beimpfung erfolgte in der Regel mit 3 ml der oben beschriebenen Vorkultur. Anschließend wurden die Kulturen unter ständigem Schütteln (240 U/min) für 4 Tage bei 28 - 30 0C inkubiert. Im Falle der Hauptkulturfermentation im mittleren bzw. großen Maßstab wurde auf das Bioreactor- System von Wave Biotech (Tageiswangen, CH) zurückgegriffen. Im Einzelnen wurden 1000 bzw. 10000 ml SC5-Medium mit 30 bzw. 300 ml Vorkultur angeimpft und für 4 Tage bei 3O0C im Wavebag bei einer Wippfrequenz von 32/min inkubiert (Winkel: 10°; Luftzufuhr: 0,25 Ltr./min). Der pH-Wert der Kulturen wurde an Tag 1 bis 3 kontrolliert und soweit notwendig mit 5 N NaOH auf pH 5-6 adjustiert. Die Kulturen wurden an Tag 1 bis 3 gefüttert, und zwar mit je 1 ml 50%-iger Hefe-Extrakt Lösung und 4 ml 4 M Glucose-Lösung im Falle der 100 ml-Kulturen. Im Falle der 1000 und 10000 ml-Kulturen erfolgte entsprechend die Zugabe des 10 bzw. 100-fachen Volumens an Fütterlösungen kontinuierlich über Tag. Zur Wachstumskontrolle konnte an verschiedenen Zeitpunkten die OD60O der Kulturen bestimmt werden. Nach 4 Tagen erfolgte die Ernte der zellfreien Überstände durch Zentrifugation (15 min bei 6000 UPM im JA14-Rotor).The main culture fermentations on a small scale were carried out using 1 liter shake flasks filled with 100 ml SC5 nutrient solution. The inoculation was usually carried out with 3 ml of the preculture described above. Subsequently, the cultures with continuous shaking (240 rev / min) for 4 days at 28 were - incubated 30 0 C. In the case of the main culture fermentation on a medium or large scale, the bioreactor system of Wave Biotech (Tageiswangen, CH) was used. Specifically, 1000 and 10000 ml of SC5 medium were inoculated with 30 or 300 ml pre-culture and for 4 days at 3O 0 C in Wavebag at a bounce frequency of 32 / min incubated (angle: 10 °; air supply: 0.25 liter. / 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 correspondingly continuously over the day. For growth control the OD 60O of the cultures could be determined at different times. After 4 days, the cell-free supernatants were harvested by centrifugation (15 min at 6000 rpm in the JA14 rotor).
Beispiel 4Example 4
Reinigung von BPTI-mutl3 aus den Überständen fermentierter Hefezellen Die in der Hauptkulturfermentation hergestellten, BPTI-mutl3 enthaltenden zellfreien Überstände wurden mit IM NaOH versetzt, bis der pH-Wert 7.8 betrug. In dem Überstand vorhandene Schwebeteilchen wurden durch Zentrifugation mit 2,000 rpm bei 4°C (15 min; Beckman-Allegra 6KR) sedimentiert. Der Überstand wurde mit 1 ml/min auf eine 10 ml Trypsinagarose-Säule (Sigma-T1763) aufgetragen. Anschließend wurde die Säule mit 70 ml 50 mM Tris pH 7.8, 250 mM NaCl sowie mit 50 ml 50 mM Tris pH 7.8, 600 mM NaCl gewaschen. Danach wurde BPTI- mutl 3 mit 180 ml 50 mM KCl/ 10 mM HCl pH 2.0 eluiert. Die 2 ml Fraktionen wurden in Röhrchen aufgefangen, die jeweils 500 μl 200 raM Tris pH 7.6, 2 M NaCl zum Neutralisieren enthielten. BPTI-mutl3 enthaltende Fraktionen wurden durch den unten beschriebenen Test über die Hemmung von Trypsin identifiziert.Purification of BPTI-mutl3 from the supernatants of fermented yeast cells The cell-free supernatants containing BPTI-mutl3 prepared in the main culture fermentation were mixed with IM 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 at 1 ml / min to a 10 ml trypsin agarose column (Sigma-T1763). 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. Thereafter, BPTI-mutl 3 was eluted with 180 ml of 50 mM KCl / 10 mM HCl pH 2.0. The 2 ml fractions were collected in Collected tubes each containing 500 ul 200 raM Tris pH 7.6, 2 M NaCl to neutralize. Fractions containing BPTI-mutl3 were identified by the trypsin inhibition assay described below.
Trypsin-hemmende Fraktionen wurden gepoolt und in einem Dialyseschlauch mit einer Ausschlußgröße von 1 ,000 Dalton (Spectra/POR6) zweimal gegen je 2 Liter 50 mM Tris pH 7.5 dialysiert. Das Dialysat wurde in einer Amicon 8200 Rührzelle über eine Ultrafiltrationsmembran mit Ausschlußgröße 1 ,000 Dalton aufkonzentriert. Anschließend wurde die Proteinkonzentration mit einem Coomassie Plus Test (Pierce, 23236) nach den Angaben des Herstellers bestimmt. Die gemessene Proteinkonzentration lag typischerweise zwischen 0.1 und 6 mg/ml. Alternativ wurden die Trypsin-hemmenden Fraktionen nach der Reinigung über Trypsin- Agarose gepoolt, mit dem gleichem Volumen 0.1% TFA gemischt und auf eine Source 15 RPC-Säule aufgetragen. Die Säule wurde mit 6 ml 0.1% TFA (Puffer HPLC-A) gewaschen, und anschließend wurde BPTI-mutl 3 mit einem 25 ml Gradienten auf 50% Puffer HPLC-B (0.1% TFA, 60% Acetonitril) sowie einem weiteren 5 ml Gradienten auf 100% Puffer HPLC-B eluiert. Die BPTI- mutl3 enthaltenden Eluate wurden lyophilisiert und das Lyophilisat in 250 μl 50 mM Tris pH 7.5 pro Fraktion aufgenommen.Trypsin-inhibiting fractions were pooled and dialysed twice in a dialysis tubing with a 1,000,000 dalton cut-off size (Spectra / POR6) against each 2 liters of 50 mM Tris pH 7.5. The dialyzate was concentrated in an Amicon 8200 stirred cell over an ultrafiltration membrane of exclusion size 1, 000 daltons. Subsequently, the protein concentration was determined with a Coomassie Plus test (Pierce, 23236) according to the manufacturer's instructions. The measured protein concentration was typically between 0.1 and 6 mg / ml. Alternatively, the trypsin-inhibiting fractions were pooled after purification over trypsin agarose, mixed with the same volume of 0.1% TFA and applied to a Source 15 RPC column. The column was washed with 6 ml of 0.1% TFA (buffer HPLC-A) and then BPTI-mutl 3 with a 25 ml gradient on 50% buffer HPLC-B (0.1% TFA, 60% acetonitrile) and another 5 ml Gradients to 100% buffer HPLC-B eluted. The eluates containing BPTI-mutl3 were lyophilized and the lyophilizate was taken up in 250 μl of 50 mM Tris pH 7.5 per fraction.
Beispiel 5Example 5
Charakterisierung von BPTI-mutl3 anhand der tryptischen Peptide und desCharacterization of BPTI-mutl3 Using the Tryptic Peptides and the
Molekulargewichtes Zur Molekulargewichtsbestimmung der vorliegenden BPTI Mutante wurde das aufgereinigte Protein mit einer 0.1% TFA Lösung auf 2pmol/μl verdünnt und gleichzeitig sauer gestellt. Diese Probe wurde nach Trennung über eine GromSil 120 ODS-4 HE (3μm, 250 x 0,2mm) massenspektrometrisch analysiert. Anhand der erhaltenen mehrfach geladenen Substanzionen konnte das Molekulargewicht von BPTI mut 13 (6379.1 Dalton) mit einem delta von 0,7 Dalton eindeutig nachgewiesen werden. Molecular Weight For molecular weight determination of the present BPTI mutant, the purified protein was diluted to 2 pmol / μl with a 0.1% TFA solution and acidified at the same time. After separation, this sample was analyzed by mass spectrometry using a GromSil 120 ODS-4 HE (3 μm, 250 × 0.2 mm). The molecular weights of BPTI mut 13 (6379.1 daltons) were unambiguously demonstrated with a delta of 0.7 daltons on the basis of the multiply charged substance ions obtained.
Zur genaueren Bestimmung der Aminosäurensequenz wurde das Protein, nach Denaturierung mit Guanidinium Hydrochlorid, Reduzierung mit Dithiotreitol und Derivatisierung mit Iodacetamid, tryptisch gespalten. Die entstandenen Spaltpeptide wurden ebenfalls massenspektrometrisch analysiert und anhand der nachgewiesenen Peptidmassen sowie MS/MS-Spektren eine genaue Sequenzabdeckung des Proteins erstellt. Sequenzabdeckung BPTI mutl3For more precise determination of the amino acid sequence, the protein, after denaturation with guanidinium hydrochloride, reduction with dithiothreitol and derivatization with iodoacetamide, was cleaved by tryptic cleavage. The resulting cleavage peptides were also analyzed by mass spectrometry and, based on the proven peptide masses and MS / MS spectra, accurate sequence coverage of the protein was established. Sequence coverage BPTI mutl3
AEDFCLEPPETGPCRAAHPRYFYNAKAGSCQTFVYGGCRGNRNNFKSEEDCMRTCGGAAEDFCLEPPETGPCRAAHPRYFYNAKAGSCQTFVYGGCRGNRNNFKSEEDCMRTCGGA
[M+H]+1 1777,73: AEDFCLEPPETGPCR[M + H] +1 1777.73: AEDFCLEPPETGPCR
[M+H]+1 2310,08: AEDFCLEPPETGPCRAAHPR[M + H] +1 2310.08: AEDFCLEPPETGPCRAAHPR
[M+H]+1 3096,57: AEDFCLEPPETGPCRAAHPRYFYNAK[M + H] +1 3096.57: AEDFCLEPPETGPCRAAHPRYFYNAK
[M+H]+1 1337,59: AAHPRYFYNAK[M + H] +1 1337.59: AAHPRYFYNAK
[M+H]+1 805,38: YFYNAK[M + H] +1 805.38: YFYNAK
[M+H]+1 2249,03: YFYNAKAGSCQTFVYGGCR[M + H] +1 2249.03: YFYNAKAGSCQTFVYGGCR
[M+H]+1 1462,55: AGSCQTFVYGGCR[M + H] +1 1462.55: AGSCQTFVYGGCR
[M+H]+1 3646,75: AGSCQTFVYGGCRGNRNNFKSEEDCMRTCGGA[M + H] +1 3646.75: AGSCQTFVYGGCRGNRNNFKSEEDCMRTCGGA
Anhand der Peptide konnten alle Veränderungen in der Aminosäurensequenz nachgewiesen werden. Using the peptides, all changes in the amino acid sequence could be detected.
Beispiel 6Example 6
Bestimmung der inbibitorischen Potenz von BPTI-mutl3 gegen Trypsin, Plasmin undDetermination of the inhibitory potency of BPTI-mutl3 against trypsin, plasmin and
PlasmakallikreinPlasma kallikrein
Die inhibitorische Potenz von BPTI-mutl3 gegen die enzymatischen Aktivitäten von Trypsin, Plasmin und Plasmakallikrein wurden in biochemischen Assays in weißen 384-Loch- Mikrotiterplatten mit Hilfe von fluorogenen Substraten bestimmt. Der Assaypuffer setzte sich zusammen aus 50 mM Tris/Cl, pH 7.4, 100 mM NaCl, 5 raM CaCl2, 0.08 % (w/v) BSA. Die Testbedingungen waren im Einzelnen wie folgt:The inhibitory potency of BPTI-mutl3 against the enzymatic activities of trypsin, plasmin and plasma kallikrein were determined in biochemical assays in white 384-well microtiter plates using fluorogenic substrates. The assay buffer was composed of 50 mM Tris / Cl, pH 7.4, 100 mM NaCl, 5 mM CaCl 2 , 0.08% (w / v) BSA. The test conditions were as follows:
Figure imgf000015_0001
Figure imgf000015_0001
Je Loch wurden 10 μl einer seriellen Verdünnung von BPTI-mutl3 vorgelegt und mit 20 μl Enzym für 5 min bei RT vorinkubiert. Anschließend wurde die Reaktion durch Zugabe von je 20 μl Substrat gestartet. Die Messung erfolgte nach 60-90 min in einem Tecan Reader bei einer Anregungswellenlänge von 360 nm und einer Emissionswellenlänge von 465 nm. Dosis- Wirkungskurven und halbmaximale inhibitorische Konstanten (IC50-Werte) wurden mittels Software GraphPad Prism (Version 4.02). ermittelt. Per 10 ul of a serial dilution of BPTI-mutl3 were placed and pre-incubated with 20 ul 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.
Beispiel 7Example 7
Inhibition der Fibrinolyse durch BPTI-mutl3Inhibition of fibrinolysis by BPTI-mutl3
Die Wirkung von BPTI-mutl 3 wurde in einem in vitro Fibrinolysemodell getestet und mit derThe effect of BPTI-mutl 3 was tested in an in vitro fibrinolytic model and compared with the
Wirkung von Trasylol (Aprotinin) verglichen. Humanes Citratplasma wurde mit 0.13 pM Tissue factor (TF) und 164 U/ml Gewebe-Plasminogenaktivator (tPA) sowie mit BPTI-mutl3 oderEffect of trasylol (aprotinin) compared. Human citrated plasma was supplemented with 0.13 pM tissue factor (TF) and 164 U / ml tissue plasminogen activator (tPA) as well as BPTI mutl3 or
Aprotinin in verschiedenen Konzentrationen (0.1 μM bis 10 μM) versetzt und 40 min bei 37°C inkubiert. Als Kontrolle wurde anstelle der Kunitz-Domänen physiologische Kochsalzlösung verwendet. Die Clot-Bildung durch TF und die Clot-Lyse durch tPA wurden durch Messungen der optischen Dichte (OD 405 nm) mit einem Tecan Safire bestimmt. Fibrinolyse wurde definiert als die relative Abnahme der OD nach Clot-Bildung . Die Hemmung der relativen Abnahme der OD bedeutet anti-fibrinolytische AktivitätAprotinin in different concentrations (0.1 uM to 10 uM) and incubated for 40 min at 37 ° C. 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. The inhibition of the relative decrease in OD means anti-fibrinolytic activity
Beispiel 8Example 8
Inhibition der Koagulation durch BPTI-mutl3 Die Wirkung von BPTI-mutl3 wurde in einem in vitro-Koagulationsmodell getestet und mit der Wirkung von Trasylol (Aprotinin) verglichen. Humanes Citratplasma wurde mit 12 mM CaCl2 zur Gerinnungsauslösung sowie mit BPTI-mutl3 oder Aprotinin in verschiedenen Konzentrationen (0.1 μM bis 10 μM) versetzt. Als Kontrolle wurde anstelle der Kunitz-Domänen physiologische Kochsalzlösung verwendet. Während der Inkubation für 90 min bei 370C wurde der Anstieg der OD bei 405 nm als Maß für die Koagulation bestimmt. Daraus wurde die halbmaximale Koagulationszeit berechnet. Eine Verlängerung der halbmaximalen Koagulationszeit bedeutet Inhibition der Koagulation.Inhibition of Coagulation by BPTI-mutl3 The effect of BPTI-mutl3 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 BPTI-mutl3 or aprotinin in various concentrations (0.1 μM to 10 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.
Beispiel 9 Kreuzreaktivitätsuntersuchung mittels kompetetivem ELISA (Antigenizitätsanalyse)Example 9 Cross Reactivity Examination by Competitive ELISA (Antigenicity Analysis)
Zwecks Evaluierung der Kreuzreaktivität der beschriebenen BPTI-Varianten mit Seren aus Aprotinin-behandelten Patienten wurde ein kompetetiver ELISA etabliert. Hierzu wurde ein kommerziell erhältlicher ELISA für den Nachweis von anti-Aprotinin-Antikörpern in Humanserum (CellTrend, Luckenwalde) so modifiziert, dass mit hoher Sensitivität die Konkurrenz der Varianten und Aprotinin um die Bindung an anti-Aprotinin-Antikörper nachgewiesen werden konnte. Die anti-Aprotinin-Antikörper-haltigen Antiseren stammten aus Aprotinin behandelten Patienten, die einen hohen Antikörpertiter gegen das Protein entwickelt hatten. Im Einzelnen wurden die Antiseren im Verdünnungspuffer zusammen mit Aprotinin bzw. der zu testenden BPTI-Variante bei Raumtemperatur für 1 h unter leichtem Schütteln in einem Gesamtvolumen von je 300 μl vorinkubiert. Hierbei kamen in der Regel je nach verwendetem Antiserum Verdünnungen von 1 :3000 bis 1 :7500 zum Einsatz. Jedes zu untersuchende Protein wurde bei drei verschiedenenen Konzentrationen getestet (1, 10, 100 nM). Eine Leerkontrolle ohne Aprotinin oder BPTI-Variante wurde als Referenz mitgefühlt. Nach der Vorinkubation erfolgte der Transfer des Ansatzes in je ein mit Aprotinin vorbeschichtetetes Well der im Test-Kit enthaltenen Mikrotiter-Module. Nach einer weiteren Inkubationszeit von 1 h bei Raumtemperatur wurde jedes Well gemäß Herstellerangaben gewaschen und anschließend eine Farbreaktion mit Hilfe des mitgelieferten Peroxidase-gekoppelten Sekundärantikörpers erzeugt. Die Messung der optischen Dichte erfolgte in einem Tecan Reader bei 450 nm (Referenzwellenlänge 620 nm). Zur Auswertung wurden die Messwerte für jede getestete Proteinkonzentration zum Referenzleerwert (100%) ins prozentuale Verhältnis gesetzt. Varianten mit im Verhältnis zum Aprotinin verringerten Kreuzreaktivitäten zu den verwendeten Antiseren wiesen typischerweise ELISA-Restsignale von > 20 % bei 100 nM Protein auf (Kreuzreaktivitäts-Score: 1). Aprotinin und Varianten mit ähnlicher oder erhöhter Kreuzreaktivität zeichneten sich dagegen durch Restsignale von < 20 % bei 100 nM Protein aus (Kreuzreaktivitätsscore: 0).In order to evaluate the cross-reactivity of the described BPTI variants with sera from aprotinin-treated patients, a competitive ELISA was established. For this purpose, a commercially available ELISA for the detection of anti-aprotinin antibodies in human serum (CellTrend, Luckenwalde) was modified so that the competition of the variants and aprotinin for binding to anti-aprotinin antibodies could be detected with high sensitivity. The anti-aprotinin antibody-containing antisera were from aprotinin-treated patients who had developed a high antibody titre to the protein. In detail, the antisera in the dilution buffer together with aprotinin or the BPTI variant to be tested were preincubated at room temperature for 1 h with gentle shaking in a total volume of 300 μl each. This usually came depending on the used Antiserum dilutions of 1: 3000 to 1: 7500 are used. Each protein to be tested was tested at three different concentrations (1, 10, 100 nM). An empty control without aprotinin or BPTI variant was felt as a reference. After preincubation, the batch was transferred to a well precoated with aprotinin well of the microtiter modules contained in the test kit. After a further incubation period of 1 h at room temperature, each well was washed according to the manufacturer's instructions and then a color reaction was generated with the aid of the peroxidase-coupled secondary antibody supplied. The optical density was measured in a Tecan Reader at 450 nm (reference wavelength 620 nm). For evaluation, the measured values for each protein concentration tested were set to the reference blank value (100%) in the percentage ratio. Variants with cross-reactivities reduced relative to aprotinin to the antisera used were typically> 20% residual ELISA at 100 nM protein (cross-reactivity score: 1). By contrast, aprotinin and variants with similar or increased cross-reactivity were characterized by residual signals of <20% at 100 nM protein (cross-reactivity score: 0).
Beispiel 10Example 10
T-Zell-Epitop Profilierung (Immunogenizitätsanalyse)T cell epitope profiling (immunogenicity analysis)
Die Epibase®-Platform (Desmet [1992], Desmet [1997], Desmet [2002], Desmet [2005]), vonThe Epibase® Platform (Desmet [1992], Desmet [1997], Desmet [2002], Desmet [2005]), by
AlgoNomics NV, Belgien, wurde eingesetzt um Aprotinin und Aprotinin-Varianten auf HLA Klasse II Epitope, auch als TH-Epitope bezeichnet, zu untersuchen.AlgoNomics NV, Belgium, was used to investigate aprotinin and aprotinin variants on HLA class II epitopes, also referred to as T H epitopes.
Kurz zusammengefaßt analysiert die Epibase®-Platform alle möglichen 10 Aminosäurereste langen Peptidteilstücke einer zu untersuchenden Zielsequenz auf Bindung gegenüber 48 HLA Klasse II Rezeptoren (Allotypen sind 20 x DRBl , 7 x DRB3/4/5, 14 x DQ und 7 x DP). Es wird die freie Bindungsenergie berechnet und darüber eine Dissoziationskonstante (Kd) bestimmt. Die jeweiligen Peptide werden klassifiziert als starke (S), mittlere (M) und schwach bzw. nicht (N) Binder. Folgende Grenzwerte wurden verwendet: S: starke Binder, Kd < 0,1 μM; M: mittlere Binder, 0,1 μM < IQ < 0,8 μM; N: schwache bzw. nicht Binder, 0,8 μM < Kd. In der humoralen Antwort, die gegenüber einem Antigen ausgelöst wird, wird die zu beobachtende TH-Zellen Aktivierung / Proliferation generell als DRBl spezifisch interpretiert. Es kann eine Beteiligung der DRB3/4/5, DQ und DP allerdings nicht ausgeschlossen werden. Aufgrund der geringeren Expressionsrate dieser Gene im Vergleich zu DRBl , werden ausschließlich starke Binder für DRB3/4/5, DQ und DP in die Betrachtung der kritischen Epitope mit einbezogen. Zu den kritischen Epitopen werden daher starke Binder gegenüber den DRBl , DRB3/4/5, DQ und DP und zusätzlich mittlere Binder gegenüber DRBl gezählt. Zur Darstellung der Immunogenizität wird die Anzahl der bindenden Peptidteilstücke einer Zielsequenz gegenüber HLA Klasse II Rezeptoren der DRBl, DRB3/4/5, DQ und DP Gene gezählt. Peptide, die an mehrere Allotypen der gleichen Gruppe binden, werden nur einmal gezählt.Briefly, the Epibase® platform analyzes all possible 10 amino acid residue long peptide portions of a target sequence to be tested for binding to 48 HLA class II receptors (allotypes are 20x DRB1, 7x DRB3 / 4/5, 14x DQ and 7x DP). The free binding energy is calculated and a dissociation constant (K d ) is determined. The respective peptides are classified as strong (S), middle (M) and weak or non (N) binders. The following limits were used: S: strong binders, K d <0.1 μM; M: middle binders, 0.1 μM <IQ <0.8 μM; N: weak or not binder, 0.8 μM <K d . In the humoral response elicited against an antigen, the observed T H cell activation / proliferation is generally interpreted as DRBI specific. However, participation of the DRB3 / 4/5, DQ and DP can not be ruled out. Due to the lower expression rate of these genes compared to DRBl, only strong binders for DRB3 / 4/5, DQ and DP are included in the consideration of the critical epitopes. The critical epitopes are therefore strong binders compared to the DRBl, DRB3 / 4/5, DQ and DP and additionally middle binders against DRBl counted. To illustrate the immunogenicity, the number of binding peptide portions of a target sequence to HLA class II receptors of DRBl, DRB3 / 4/5, DQ and DP genes counted. Peptides that bind to multiple allotypes of the same group are counted only once.
Beispiel 11 MigrationsassayExample 11 Migration Assay
Humane koronare arterielle vaskuläre glatte Muskelzellen (hCAVSMC, 1 ,5x105 Zellen/well) (TEBU, Offenbach, Deutschland) werden in einer 6-Well Platte ausgesät und über 48 h in M 231 Medium (Wachstumsmedium) (TEBU, Offenbach, Deutschland) bei 37°C/ 5% Kohlendioxid kultiviert. Die Platten werden zuvor mit Vitronectin (50 ng/cm2) (Gibco/Invitrogen, Karlsruhe, Deutschland) beschichtet. Nach der Inkubationszeit wird eine Hälfte des konfluenten Zellmonolayers entfernt. Im zellfreien Bereich des Wells bleibt ca. 50% der Vitronectinbeschichtung erhalten.Human coronary arterial vascular smooth muscle cells (hCAVSMC, 1, 5x105 cells / well) (TEBU, Offenbach, Germany) are seeded in a 6-well plate and for 48 h in M 231 medium (growth medium) (TEBU, Offenbach, Germany) at 37 ° C / 5% carbon dioxide cultivated. The plates are previously coated with vitronectin (50 ng / cm 2) (Gibco / Invitrogen, Karlsruhe, Germany). After the incubation period, one half of the confluent cell monolayer is removed. In the cell-free area of the well about 50% of the vitronectin coating is retained.
Das Wachstumsmedium wird durch das Testmedium MCDB-131/0,2% BSA (molecular cellular developmental biology (MCDB); Basalmedium (BSA)) (Gibco/Invitrogen, Karlsruhe, Deutschland) ersetzt und die Zellen werden mit 1OnM PDGF-BB ((R&D Systems, Wiesbaden- Nordenstadt, Deutschland) stimuliert.The growth medium is replaced by the test medium MCDB-131 / 0.2% BSA (Molecular Cellular Developmental Biology (MCDB); Basal Medium (BSA)) (Gibco / Invitrogen, Karlsruhe, Germany) and the cells are replaced with 1OnM PDGF-BB (( R & D Systems, Wiesbaden-Nordenstadt, Germany).
Die Testsubstanzen werden dann in den angegebenen Konzentrationen zugesetzt. Nach 24- und 48-stündiger Inkubation wird die Migrationsstrecke der Zellen in die freie Wellfläche mikroskopisch bestimmt. Jeder Messpunkt repräsentiert einen Mittelwert aus vier gemessenen Regionen und mindestens drei unabhängige Experimenten wurden durchgeführt.The test substances are then added in the indicated concentrations. After 24 and 48 hours of incubation, the migration distance of the cells into the free corrugation area is determined microscopically. Each measurement point represents an average of four measured regions and at least three independent experiments were performed.
Die Migrationstrecken werden in Korrelation zu der PDGF-induzierten Migrationstrecke (=100%) berechnet.The migration distances are calculated in correlation to the PDGF-induced migration distance (= 100%).
Beispiel 12Example 12
Chemotaxis-AssayChemotaxis assay
Neutrophile werden nach Standardmethoden aus Blut isoliert.Neutrophils are isolated from blood by standard methods.
Die Chemotaxis der Neutrophile wird in einem two chamber System durchgeführt. Auf die benutzte Membran (3-μm Porengröße, Polycarbonate, Fa. Falcon) wird ein HUVEC Monolayer über 24h kultiviert. 1x105 Neutrophile in RPMI 1640 Medium, die zuvor mit einem Fluoreszenzfarbstoff beladen wurden, werden in die obere Kammer gegeben. Die untere Kammer enthält variierende Konzentrationen an Stimulus oder eine konstante Stimuluskonzentration (IL-8: 5nM oder C5a: 1OnM) und variierende Konzentrationen der Testsubstanz. Die zu untersuchenden Substanzen befinden sich in beiden Kammer. Der Testansatz wird für 45min bei 37°C und 5% CO2 inkubiert. Nach der Inkubation werden die Zellen bestimmt, die in die untere Kammer gewandert sind (Fluoreszenzmessung, Zählung). The chemotaxis of neutrophils is performed in a two chamber system. On the used membrane (3-micron pore size, polycarbonate, Fa. Falcon) a HUVEC monolayer is cultured for 24 h. 1x105 Neutrophils in RPMI 1640 medium, previously loaded with a fluorescent dye, are placed in the upper chamber. The lower chamber contains varying concentrations of stimulus or constant stimulus concentration (IL-8: 5nM or C5a: 1OnM) and varying concentrations of the test substance. The substances to be investigated are in both chambers. The assay is incubated for 45 min at 37 ° C and 5% CO2. After incubation, the cells which have migrated to the lower chamber are determined (fluorescence measurement, counting).
Tabelle 1:Table 1:
Numerierung der Aminosäurereste einer Kunitz-Domäne nach Aprotinin-Schema:Numbering of the amino acid residues of a Kunitz domain according to aprotinin scheme:
1 2 31 2 3
1234567890123456789012345678901234567890123456789012345678 xxxxCxxxxxxxxCxxxxxxxxxxxxxxxCxxxxxxxCxxxxxxxxxxxxCxxxCxxx 10XxxxCxxxxxxxxCxxxxxxxxxxxxxxxCxxxxxxxCxxxxxxxxxxxxCxxxCxxx 10244567890123456789012345678901234567890123456789012345678
9090
Aminosäurereste 1 , 2, 3, 4 können fehlen 15 Aminosäurereste 56, 57, 58 können fehlenAmino acid residues 1, 2, 3, 4 may be missing 15 amino acid residues 56, 57, 58 may be missing
Zusätzliche Insertionen und Deletionen zwischen Aminosäureresten 5 und 55 sind möglich Additional insertions and deletions between amino acid residues 5 and 55 are possible
Tabelle 2:Table 2:
Sequenzen einiger natürlicher Kunitz-DomänenSequences of some natural Kunitz domains
^o
Figure imgf000021_0001
^ o
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000022_0001
Tabelle 3:Table 3:
Sequenzen einiger nicht-natürlicher Kunitz-Domänen
Figure imgf000023_0001
Sequences of some non-natural Kunitz domains
Figure imgf000023_0001
Tabelle 4:Table 4:
IC50- Werte für die Hemmung von humanem Trypsin, Plasmin und Plasmakallikrein und Kreuzreaktivität der BPTI- Varianten 1 bis 16:IC50 values for the inhibition of human trypsin, plasmin and plasma kallikrein and cross-reactivity of BPTI variants 1 to 16:
K* K*
Figure imgf000024_0001
K * K *
Figure imgf000024_0001
K*
Figure imgf000025_0001
K *
Figure imgf000025_0001
': Mittelwerte aus mind. 2 Experimenten': Mean values from at least 2 experiments
2: Kreuzreaktivitätsscore 0: < 20% Restsignal im ELISA bei 100 nM Protein; Kreuzreaktivitätsscore 1 : > 20% Restsignal im ELISA bei 100 nM Protein 2 : cross reactivity score 0: <20% residual signal in ELISA at 100 nM protein; Cross-reactivity score 1:> 20% residual signal in ELISA at 100 nM protein
Tabelle 5:Table 5:
TH-Epitop-Anzahl pro HLA-Gen für Aprotinin und Aprotinin-Varianten. Peptide, die an mehrere HLAs desselben Genes (DRB1 , DRB3/4/5, DQ und DP) binden, werden einmal gezählt (Definitionen siehe Ausführungsbeispiel)T H epitope number per HLA gene for aprotinin and aprotinin variants. Peptides which bind to several HLAs of the same gene (DRB1, DRB3 / 4/5, DQ and DP) are counted once (for definitions, see embodiment)
Figure imgf000026_0001
Figure imgf000026_0001
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AbkürzungenAbbreviations
AUC Fläche unter der KurveAUC area under the curve
BPTI Bovine pancreatic trypsin inhibitorBPTI bovine pancreatic trypsin inhibitor
DIC Disseminierte intravasale GerinnungDIC Disseminated intravascular coagulation
DNA Deoxyribonukl einsäureDNA deoxyribonucleic acid
HPLC High Performance liquid chromatography hTFPI human tissue factor pathway inhibitor 1 hTFPI Dl human tissue factor pathway inhibitor 1 Kunitz-Domäne 1HPLC High Performance Liquid Chromatography hTFPI human tissue factor pathway inhibitor 1 hTFPI Dl human tissue factor pathway inhibitor 1 Kunitz domain 1
HLA human leukocyte antigenHLA human leukocyte antigen
IC50 Inhibitorkonzentration bei 50% Hemmung der Enzymaktivität kDa KilodaltonIC50 inhibitor concentration at 50% inhibition of the enzyme activity kDa kilodaltons
M molar (mol pro Liter) min MinuteM molar (mol per liter) min
MOF Multi-OrganversagenMOF multi-organ failure
OD AbsorptionOD absorption
PEG PolyethylenglykolPEG polyethylene glycol
PCR Polymerase-Kettenreaktion rpm Umdrehungen pro MinutePCR polymerase chain reaction rpm revolutions per minute
TFA TrifluoressigsäureTFA trifluoroacetic acid
TPA tissue Plasminogen activatorTPA tissue plasminogen activator
U Unit (Einheit der Enzymaktivität) U unit (unit of enzyme activity)

Claims

Patentansprüche claims
1. Aprotinin Varianten der allgemeinen Formel1. Aprotinin variants of the general formula
X1X2DFCLEPPX1OTGPCX15XI6XI 7XI 8XI9RYFYNAKAGX29CQTFVYGGCRX4OX4IRNNFK SX48EDCMRTCGGAX 1 X 2 DFCLEPPX 1O TGPCX 15 X I6 X I 7 X I 8 X I9 RYFYNAKAGX 29 CQTFVYGGCRX 4O X 4I RNNFK SX 48 EDCMRTCGGA
mit mindestens einer Mutation in der Position X4g gegenüber der natürlichen Sequenz.with at least one mutation in position X 4 g with respect to the natural sequence.
2. Aprotinin Varianten gemäß Anspruch 1 mit einer zusätzlichen Mutation in der Position X1O gegenüber der natürlichen Sequenz.2. Aprotinin variants according to claim 1 with an additional mutation in the position X 1 O compared to the natural sequence.
3. Aprotinin Varianten gemäß Anspruch 1 mit einer zusätzlichen Mutation in der Position X40 gegenüber der natürlichen Sequenz.3. Aprotinin variants according to claim 1 with an additional mutation in the X 40 position relative to the natural sequence.
4. Aprotinin Varianten gemäß Anspruch 1 mit einer zusätzlichen Mutation in der Position X4] gegenüber der natürlichen Sequenz.4. Aprotinin variants according to claim 1 with an additional mutation in the position X 4 ] compared to the natural sequence.
5. Aprotinin Varianten gemäß Anspruch 1 ausgewählt aus der Gruppe Seq. Nr. 2 (BPTI-mutl), Seq. Nr. 3 (BPTI-mut2), Seq. Nr. 4 (BPTI-mut3), Seq. Nr. 5 (BPTI-mut4), Seq. Nr. 6 (BPTI- mut5), Seq. Nr. 10 (BPTI-mut9), Seq. Nr. 11 (BPTI-mutlO), Seq. Nr. 12 (BPTI-mutl 1), Seq. Nr. 13 (BPTI-mutl2) und Seq. Nr. 14 (BPTI-mutl3). 5. Aprotinin variants according to claim 1 selected from the group Seq. No. 2 (BPTI-mutl), Seq. No. 3 (BPTI-mut2), Seq. No. 4 (BPTI-mut3), Seq. No. 5 (BPTI-mut4), Seq. No. 6 (BPTI-mut5), Seq. No. 10 (BPTI-mut9), Seq. No. 11 (BPTI-mutlO), Seq. No. 12 (BPTI-mutl 1), Seq. No. 13 (BPTI-mutl2) and Seq. No. 14 (BPTI-mutl3).
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* Cited by examiner, † Cited by third party
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