MXPA97005605A - Aprotinin variants with improved properties - Google Patents

Abstract

The present invention relates to variants of aprotinin with improved inhibitory properties of enzymes, immunological and pharmacokinetics and their production.

Description

Aprotinin variants with improved properties DESCRIPTION OF THE INVENTION The present invention relates to variants of aprotinin with inhibitory properties of improved enzymes, immunological and pharmacokinetics and their production. Aprotinin, which is also referred to as bovine pancreatic trypsin inhibitor (ITPB), belongs to the Kunitz serine protease inhibitor family. * The spectrum of serine proteases susceptible to inhibition comprises, for example, trypsin, chymotrypsin, plasmin. and plasma kallikrein (W. Gebhard, H. Tschesche and H. Fritz, Proteinase Inhibitors, Barrett and Salvesen (eds.), Elsevier Science Publ. BV 375-387, 1986). Aprotinin is composed of 58 amino acids. The three-dimensional structure of the protein was elucidated with the aid of structural analysis by X-ray and NMR spectroscopy (Loda er et al., J. Mol. Biol. 198 (3), 469-480, 1987; Wagner et al., J. Mol. Biol. 196, (1), 227-231, 1987, Berndt et al., Biochemistry 32 (17), 4564-4570, 1993). Originally, the native aprotinin is used for the treatment of pancreatitis, under the tradename Trasylol. "Trasylol" is used today in cardiac surgery REF: 25147, after clinical studies have shown that a treatment with aprotinin decreases The need for transfusions in such operations is significant and leads to a reduction of subsequent bleeding (D. Royston, J. Cardiothorac, Vasc Anesth 6, 76-100, 1992). It could be demonstrated that the exchange of the amino acid in position 15, determinant of the inhibitory specificity, leads to valuable aprptinin variants with improved inhibitory properties (DE-PS 3 339 693). According to the amino acid introduced, potent inhibitors can be generated in this way, which inhibit, for example, the elastase of the pancreas or leukocytes. It could further be demonstrated that the inhibitory properties of aprotinin and of the variants originated by the exchange at position 15 are also determined by other amino acid residues in the region of contact between the target protease to be inhibited and the inhibitory molecule. To these belong, above all, the additional amino acid residues at positions 14, 16, 17, 18, 19, 34, 38 and 39. By way of example, variants of aprotinin with improved properties by the exchange of one or more of these amino acid residues within the contact environment were described, inter alia, in the following patent applications: WO 89 / 01968, WO 89/10374, EP 0 307 592, EP 683 229.

Interestingly, the pharmacokinetic properties of aprotinin and its variants could be improved by exchanges of amino acids that determine the physical-chemical properties of the substance. Thus, by reducing the net positive charge of the molecule, it is possible to significantly reduce the attachment to the kidney. Such variants were described in the patent application WO 92/06111. For reasons of better industrial productivity, it is favorable to undertake in certain cases a modification at the N-terminal end of the inhibitor molecule. Such modifications can be shortenings or elongations or N-terminal deletions of one or several amino acids. In EP 419 878, variants of modified aprotinin in the N-terminal position were described. Aprotinin variants according to the invention The aprotinin variants described in the present invention are distinguished by the following characteristics: 1. Exchange of one or several amino acids in the active center of the molecule for the improvement of activity properties. 2. Exchange of amino acids for the reduction of positive net charge in order to improve the immunological and pharmacokinetic properties. 3. Modification of the N-terminal amino acid sequence for reasons of industrial productivity.

The aprotinin variants that contain only one of the mentioned characteristics respectively were described in the patent applications cited above. The aprothmine variants according to the invention now combine two or three of the characteristics mentioned above in their molecular structure. In the illustration is represented, by way of example, the amino acid sequence for some variants. However, the aprotinin variants according to the invention are not restricted to the examples mentioned in the illustration 1. The variants of the aprotinin according to the invention also include variants with the N-terminal elongation Ala (-2) -Gln (- l), with the native amino acid residue proline in position 2, with the exchange of other amino acids carrying a positive charge against neutral or negatively charged amino acids, or with the exchange of other neutral amino acids against negatively charged amino acids. With this, the choice of the exchange of amino acid residues follows the principle of originating a substance that at a physiological pH value has a reduced net positive charge, preferably in the range of +2 to -2. The aforementioned changes in the amino acid sequence, including elongation or N-terminal deletions or deletions, can be used together in any combination. Therefore, all the compounds exhibiting a combination of the above-mentioned characteristics and having a net positive charge reduced to the physiological pH value belong to the aprotinin variants according to the invention. Surprisingly, it was shown that the combination of two or three of the mentioned characteristics not only leads to the obtaining, but even, partially, to the strengthening of the expression of the individual characteristics. In addition, significant new properties of the substance could be originated which relate, for example, to the immunological, pharmacokinetic and surface-binding properties. The -new variants show a diminished reaction with human and rabbit polyclonal antisera that originated with the use of aprotinin. It was also found that the new variants have a clearly diminished immunogenic behavior in comparison with aprotinin, that is, they trigger a diminished immune response. It could further be demonstrated that the variants according to the invention induce a decreased histamine release by blood cells compared to aprotinin. In addition, the new variants show a clearly decreased renal accumulation compared with aprotinin. Kinetic-enzymatic inhibition constants (Ki values) have been surprisingly improved despite the large number of changes in the skeleton of the molecule versus previous variants of the molecule.

The present invention therefore relates to aprotinin variants with a net charge of +3 to -3 at pH 7 and with the amino acids Arg 15 or Arg 15-Ala 17. Those with a net charge of +2 to -2 are preferred, with preference those with +1 to -1. These aprotinin variants are suitable for the inhibition of plasma kallikrein, tissue kallikrein and plasmin. In addition, these aprotinin variants may possess a modified N-terminal sequence. With this, aprotinin malfunctioners are alluded to with an N-terminal elongation or shortening or with amino acids deleted in the N-terminal position. Preferred are aprotinin variants of the group DesPro2-Serl0-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin, DesPro2-Serl0-Argl5-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin, DesPro2-Serl0-Argl5 -Serl7-Asp24-Thr26- Glu31-Asn41-Glu53-aprotinin, DesPro2-Serl0-Argl5-Alal7-Thr26-Glu31-Asn41-Glu53-aprinthin and DesPro2-SerlO-Argl5-Alal7-As? 24-Thr26-Asn41-Glu53 -a? rotinina. These aprotinin variants can also carry the amino acid proline in position 2. The invention also relates to medicaments containing one or more of these aprotinin variants. The described novel type protease inhibitors are suitable for the treatment of disease states in which they are reached, also as a consequence of complex surgical procedures such as, for example, in cardiac surgery or in alloartroplastic replacement of joints in transplant medicine. , to the activation of the plasmatic enzymatic system by extensive or intensive blood contact with foreign surfaces. Inhibitors reduce blood loss in operations that are linked to a high risk of bleeding (eg, heart surgery, knee and joint surgery). They are suitable for therapy in shock, polytrauma and cranial and cerebral trauma, septicemia, disseminated intravasal coagulopathy (CID), multiple organ failure, inflammatory diseases with the participation of the kallikrein system such as rheumatic diseases of joints and asthma. They prevent the invasive growth of tumors and metastasis by inhibiting plasmin. They are also suitable for the treatment of pains and edema by inhibiting the synthesis of bradykinin as well as for the treatment of apoplexy. They are also usable in dialysis therapy and artificial organs to eliminate inflammation and coagulation and to reduce the risk of bleeding. Obtaining the aprotinin variants according to the invention To obtain the aprotinin variants according to the invention, it is advantageous to use genetic engineering techniques. For this, the genetic information for the synthesis of the aprotinin variant respectively considered is introduced, by current methods of molecular biology, into a suitable microbial expression organism. The recombinant microorganism is fermented; the heterologous genetic information is brought to expression by choosing suitable conditions. The expressed aprotinin variant is then obtained from the culture broth. Suitable host organisms for the production of the aprotinin variants according to the invention can be bacteria, yeasts or fungi. The expression can be intracellular or extracellular, using appropriate secretory systems. The aprotinin variant can be expressed correctly processed or fused to a peptide or protein. Suitable systems for the expression of aprotinin variants were described in patent applications EP 683 229, WO 89/02463, WO 90/10075 and in several other patent applications already mentioned above. Methods for carrying out the invention Enzymes The enzymes used (restriction endonucleases, calf intestine alkaline phosphatase, T4 polynucleotide kinase and T4 DNA ligase) were purchased from Boehringer Mannheim and GIBCO-BRL and were used according to the requirements of maker. Molecular biology techniques Routine cloning works as, for example, the isolation of plasmid DNA from E. coli (called miniprep) and the transformation of E. coli with plasmid DNA were carried out according to Sambrook et al. (Molecular Cloning, Cold Spring Harbor, 1989). The E. coli strain DH5a (GIBCO / BRL) was used as the host organism for the transformations. For the isolation of larger amounts of plasmid DNA, Qiagen-tips (Qiagen) were used. The extraction of DNA fragments from agarose gels was carried out with the help of Jetsorb, according to the manufacturer's instructions (Genomed). Oligonucleotides for "site-directed mutagenesis" and primers for PCR and sequencing reactions were obtained with the "380 A DNA synthesizer" from Applied Biosystems. The mutagenesis assays were carried out according to a procedure by Deng and Nickoloff (Deng et al., Anal. Biochem 200. 81-88, 1992) using a kit from the Pharmacia Biotech company ("Unique Site Elimination Mutagenesis" ). All vector constructs and mutagenesis experiments were checked by Taq Cycle sequencing of DNA with fluorescently labeled terminators in an "ABI 373 A Sequencer" (Applied Biosystems).

Transformation of Saccharomyces cerevisiae Yeast cells were placed, for example strain JC3'-. 4D (MATa, ura3-52, suc2) in 10 ml of YEPD (2% of glucose, 2% of peptone, 1% of Difco yeast extract) and harvested at a DOβoo- .. of 0.16 to 0 , 8. The cells were washed with 5 ml of solution A (sorbitol, IM, Bimocin, pH 8.35, 3% ethylene glycol), resuspended in 0.2 ml of solution A and stored at -70 ° C. Plasmid DNA (5 μg) and carrier DNA (50 μg herring sperm DNA) were added to the frozen cells. The cells were then thawed by shaking at 37 &C for 5 minutes. After the addition of 1.5 ml of solution B (40% PEG 1000; 200 mM Bicin, pH 8.35), the cells were incubated at 30 ° C for 60 minutes, washed with 1.5 ml of solution C ( CINa 0.15 M; Biquin 10 mM, pH 8.35) and resuspended in 100 μl of solution C. The plating was carried out on a selective medium with 2% agar. Transformants were obtained after incubation at 302C for 3 days. Nutrient media for fermentation 1. SD2 medium: Bacto Yeast Nitrogen base 6.7 g / l Glucose * 20 g / l P04H3K 6.7 g / l pH 6.0. Medium SC5: Glucose '20 g / l Difco yeast extract 20 g / l PO «H2K 6.7 g / l S04 (NH4) 2 2.0 g / l S04Mg x 7 H.0 1.0 g / l Solution of trace elements SL4 1.0 ral / 1 pH 6.0 Solution of trace elements SL4: Titriplex III 5 g / l SO «Zn x 7 H.0 0.1 g / l Cl.Mn x 4 H.0 0.03 g / l ClaCu x 2 HJO 0.01 g / l Cl.Ni x 6 H.0 0.02 g / l Mo0Na_ x 2 H20 0.03 g / l * = Separate autoclave 3. Fermentation medium: Glucose * 2.0 g / l Soy peptone 25 g / l Thiamine Chloride 5.1 mg / 1 Inosite 20 mg / 1 Trace element solution 3 ml / 1 Vitamin solution 3 ml / 1 pH 5.5 Nutrient solution: Glucose * 530 g / l S04 (NH4) 2 5.0 g / l Thiamine chloride 13 mg / 1 Inosite 70 mg / 1 Trace solution 6.8 ml / 1 Vitamin solution 6.8 ml / 1 Trace element solution: CljCa x 2 HJO 1.0 g / l C1H conc. 100 ml / 1 * = Autoclave separately Vitamin solution: Riboflavin 0.42 g / l Ca pantothenate 5.9 g / l Nicotinic acid 6.1 g / l Pyridoxine hydrochloride 1.7 g / l Biotin 0.06 g / l Folic acid 0.04 g / l Obtaining canned food 1% was inoculated with an original canister, 200 ml of SD-2 medium in a 1-liter Erlenmeyer flask. The culture was incubated for 72 hours at 28 ° C over the shaking table (260 rpm). After this, 2 ml canisters were filled and frozen in liquid nitrogen. Fermentation in shake flasks As a pre-culture, 1% was inoculated with a working can, 200 ml of SD2 medium in a 1 1 flask and fermented for 72 hours at 280 ° C on the shaker table (260 rpm). With the preculture 1% main cultures were inoculated (200 ml of SC5 medium in flasks of 1) and incubated for 72-96 hours at 28 &C on the shaker table. Fermentation in 10 1 Bioreactors As preculture, 1% was inoculated with a working canister, 200 ml of SD2 medium in a 1 1 flask and fermented for 72 hours at 282C on the shaker table (260 rpm). The main culture in the 10 1 fermentor was carried out for 96 hours in the form of fermentation in the fed tank. As a nutrient medium, fermentation medium was used. The initial volume was 7 1. The fermenter was inoculated with 200 ml of preculture. Fermentation conditions: Temperature 282C Stirrer speed 500 rpm Aeration 10 1 / minute pH 5.5 Pressure in the headspace: 200 mbar. After a fermentation time of 7 hours, the feeding began. The feeding rate was regulated on the respiratory coefficients (CR) (CR = C02 formed / oxygen consumed). If the CR exceeded the value of 1.15, the feed rate was lowered, if it fell below the value of 1.05, the feed rate was raised. At regular intervals samples were taken from the fermentor and cell growth was determined by measuring the optical density at 700 nm. The concentration of Bay 19-8757 in the supernatant was determined by activity measurement. At the end of the fermentation the pH value was lowered to 3.0 by addition of 50% (w / v) citric acid and the fermenter was heated at 70 ° C for 10 minutes. The cells were then separated by centrifugation at 7,500 x g and the supernatant was passed for protein purification.

Materials for chemical analysis of proteins Sequence analyzes were carried out with a protein sequencer of model 473A from Applied Biosystems (Forster City, USA). The standard sequencing program was used. The sequencer, the various sequencing programs as well as the PTH detector system are described in detail in the User's Manual (471 A) (1989) Applied Biosystems, Forster City, CA 94404, USA; Reagents for the operation of the sequencer and the HPLC column for PTH detection were purchased from Applied Biosystems. The HPLC analyzes were carried out with an HPLC system HP 1090, from Hewlett Packard (D-Waldbronn). For separation, an RP-18 HPLC column (250 mm x 4.6 mm, 5 μ material, 300 pore diameter) was used.

Angstroms) from Backerbond (D-Groß Gerau). The capillary column electrophoresis model 270 A-HT was from Applied Biosystems (Forster City, CA 94404, USA). The samples were injected, usually, hydrodynamically at various time intervals. The capillary column used (50 μm x 72 cm) was from Applied Biosystems. The amino acid analyzes were carried out with an amino acid analyzer LC 3000 from Eppendorf Biotronik (D-Maintal). A slightly modified standard Biotronik separation program was used. The separation program and the operation of the analyzer are described in detail in the device manual. Molecular weights were determined with a MALDI I system from Kratos / Shiraadzu (D-Duisburg). Electrophoresis in SDS was carried out with a Pharmacia electrophoresis system (D-Freiburg). The determination of the kinetic data was carried out with the SLT microtiter plate reader (D-Crailsheim). The washing of the microtiter plates was carried out with a Dynatec washing apparatus (D-Denkendorf). The enzymes and substrates were from Calbiochem (D-Bad Soden). All other chemicals and reagents were from Merck (D-Darmstadt) or Sigma (D-Deisenhofen). The 96-well plates were purchased from Greiner. Polyclonal anti-aprotinin rabbit antibodies originated in rabbits by immunization with aprotinin. Human anti-aprotinin polyclonal antibodies came from patients who had been treated with aprotinin. Chemical analysis of proteins Analysis of the N-terminal sequence 1-3 nmol of protease inhibitor dissolved in water was loaded onto a sequencing sheet that had been pre-incubated with Polybren. The protein was sequenced with the normal fast sequencer cycle. PTH-amino acids were identified by on-line HPLC with the aid of a standard 50 pM PTH. Amino acid analysis 200 μg of protein was dissolved in 200 μl of 6 H -ClH and hydrolysed at 1662 ° C for 1 hour. About 1 nmol of the sample was placed on the amino acid analyzer. The proportion of amino acids was determined on a 5 nM standard. Electrophoresis in the with SDS The gel electrophoresis with SDS was carried out according to the conditions of Laemmli. 10 μg of the protease inhibitor was analyzed with a gel with SDS of 10-20% and visualized by staining with silver (Merril et al.). U.K. Laemmli, Nature 227, 680-685 (1970). C.R. Merril, M.L. Dunau, D. Goldmann, Anal. Biochem. 100: 201-207 (1981). Capillary electrophoresis 8 ng of the protease inhibitor was investigated by capillary column electrophoresis on a glass column (length 72 cm, inner diameter 50 μra). Conditions: current intensity 90 μA, temperature in the 25SC column, 100 nM phosphate buffer, pH 3.0, detection at 210 nm, pressure service 3 seconds. Inverted phase chromatography 5 nmol of the protease inhibitor was chromatographed on a Bakerbond RP-18 HPLC column (5 μ material, 4.6 nm x 250 mm, pore size 300 Angstroms). An acetonitrile / TFA gradient was used as the eluent. Conditions: flow 0.7 ml / minute, temperature in column 402C, detection 402C, solvent A, 0.1% TFA, solvent B, 0.1% TFA / 60% acetonitrile; gradient: 0 minutes 0% B, 10 minutes 0% B, 70 minutes 100% B, 80 minutes 0% B. Determination of molecular weights 1 μg of the protease inhibitor was analyzed with the MALDI technique. As a matrix, sinapic acid was used. The standard proteins for mass calibration were bovine insulin, cytochrome C and melittin. Protein content The protein content was determined by the BCA method. In this method, through Cu2 * ions Cu2 * ions are transformed through the present proteins, which form a complex that absorbs at 560 nm with bicinconic acid. The lyophilized protein is brought to its equilibrium moisture and dissolved in 0.9% CINa solution at a concentration of 1 mg / ml. A series of dilutions is obtained. To 50 μl of sample solution is added 1000 μl of BCA assay reagent (Pierce), the test tubes are solidly closed with a stopper and incubated at 60 ° C for exactly 30 minutes. After cooling the samples in an ice bath for 5 minutes, the measurement is carried out at a temperature of 252C and at a wavelength of 560 nm. Activity: (assay of trypsin inhibition, titrimetric) The activity is determined according to an inhibition assay F. I .P. -tripsin modified. Bay and 19-8757 inhibit the trypsin-catalyzed hydrolysis of Na-benzoyl-L-arginineethyl ester (BAEE). The carboxyl groups released in the reaction are determined by alkaline titration. The residual activity of trypsin is. a measure of the inhibitory activity of the active substance. The lyophilized protein is brought to its equilibrium moisture, dissolved in 0.9% CINa solution at a concentration of 1 mg / ml and a series of dilutions are obtained. To 1 ml of sample solution is added 2 ml of buffer (15 mM borate buffer, pH 8.0 with 200 mM Cl2Ca) and 0.8 ml of trypsin solution (2 mg / ml) and incubated at 252C for 5 minutes. minutes Then 0.2 ral of BAEE solution (6.8 mg / ml) is added and the KOH consumption is measured for 5 minutes. Determination of the cross-reaction of protease inhibitors with polyclonal anti-aprotinin antibodies of either human or a microtiter plate, were bound overnight at 2C, 0.5-10 ng of protease inhibitor or aprotinin dissolved in coupling buffer. The wells were washed 4 times with 200 μl of wash buffer each time and then 100 μl of the blocking solution was added. The plates were covered and incubated at 372C for one hour. After washing as described above, polyclonal rabbit anti-aprotinin antibodies (0.2 μg / ml in 1% BSA in PBS buffer) or polyclonal anti-human aprotinin antibodies (20 μg / ml in 1% HSA in PBS buffer). The plates were covered and incubated at 372C for one hour and then washed as described above. Then 100 μl of biotinylated anti-rabbit or anti-human antibodies (25 μl + 10 ml of 1% BSA or 1% HS in PBS buffer) were added and incubated at 37 ° C. for one hour. The plates were washed as described above and then 100 μl of streptavidin-peroxidase complex (50 μl + 10 μl of 1% BSA or 1% HSA in PBS buffer) was added to each well. Plates were covered and incubated at 37 ° C. for one hour and then washed as described above. The substrate reaction was carried out with TMB-substrate + peroxidase solution (1 + 1, 100 μl per well). The reaction was stopped after 10 minutes with 100 μl / well of 2M phosphoric acid and the absorption was measured at 450 nm (reference 570 nm). Solutions: 1. Incubation buffer: 15 nM COiNa, 35 mM COaHNa, pH 9.6.e buffers: Samples were dissolved in incubation buffer at a suitable concentration. 3. Washing solution: 0.1% Tween-20 (v / v) in PBS. 4. Blocking buffer: BSA or 3% HSA (w / v) in PBS. EXAMPLES Example 1 Preparation of a levator expression vector for the secretion of recombinant DesPro2-Serl0-Arql5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin. The DesPro2-Serl0-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin gene was used as DesPro2-Argl5-Alal7 gene, which was cloned into the pUCld vector by means of restriction enzymes. HindIII and BamHl. The resulting vector (pEM6.6.L) was subjected to the mutagenic primer A and the selection primer Scal / Mlul U.S.E. to a double-stranded mutagenesis reaction according to the U.S.E. (Pharmacia Biotech). Mutagenic primer A had the following sequence: Primer A 5'GGCTGCAGAGCTAACCGTAACAACTTCAAATCCGCGGAAGACTGCATG GAAACTTGCGC GGTGCTTAG_3_'.

This primer generates the mutations Asn41 and Glu53 in the gene of DesPro2-Argl 5-Alal7-aprotinin. The analysis of the clones was carried out by means of a restriction digestion with the enzymes Seal and Sphl. The desired sequence was also confirmed by sequencing the DNA of the clone pEM31.8.L. The other exchanges in the 5 'region of the gene (Serl0-Asp24-Thr26-Glu31) originated with the help of the PCR technique with the use of primer B and the primer "M13 24-inverse", from the DNA of the plasmid pEM31.8.L. Primer B had the following sequence: Primer B 5'TGCCTCGAGCCGCCGTCTACTGGGCCCTGCAGAGCTATCATCC TT CT TCTACGATGCAACTGCAGGCCTGTGTGAAACCTTCGTATACGGC 3 '.

The Xhol recognition sequence is underlined. The PCR mixture contained 20 ng of plasmid DNA pEM31.8.L, 20 pmol of primer "M13 24-inverse mero", 60 pmol of primer B, 200 μM of dNTPs, 1 x buffer II for PCR reaction (Perkin Elmer ), 4 mM of CljMg and 2.5 U of Taq DNA polymerase (Perkin Elmer) in a total volume of 100 μl. The conditions of the "cycle" were 3 minutes at 94 ° C, 30 cycles, respectively from 1 minute at 942 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C and a subsequent incubation of 5 minutes at 722 ° C. The PCR mixture was diluted 1: 5 and ligated with the pCRII vector (Invitrogen). E.coli DH5a cells were transformed with the ligation mixture. Positive clones were identified after restriction digestion with the Xhol and BamHl enzymes and several clones were sequenced.

The clone pES9.10.L contained the desired sequence and was used for subsequent work. An E. coli / yeast seesaw vector (e.g. pA202) was employed for the construction of a yeast secretion vector in which the sequence DesPro2-SerlO-Argl5-AlaI7-Asp24-Thr26-Glu31-Asn41-Glu53-a? Rotinin has been directly linked to the pre-pro sequence of the alpha factor of the yeast. The vector pA202 carries an ampicillin resistance gene (bla) and a URA3 gene as a selectable marker gene for E. coli and yeast. Other essential elements of the vector are the point of origin of the replication (ori) of Col El and 2μ. The REP3 site is also located in this region. An EcoRI-HindIII fragment, 1,200 base pairs in length, carries the MFal promoter and the N-terminal pre-pro sequence of the yeast alpha factor precursor protein (Kurjan and Herskowitz, Cell 30, 933-943, 1982 ). By introducing a modified cDNA of DesPro2-Argl5-aprotinin as a HlndlII-BamHI fragment, the recognition site for the KexII protease ("Lys-Arg") was retrieved within the pre-pro sequence of alpha factor (Document EP 0 419 878). At the 3 'end of the DesPro2-Arg-15-aprotinin sequence, the vector carries a BamHI-SalI fragment of the yeast gene URA3, which in this position acts as a termination signal for transcription (Yarger et al. Mol. Cell Biol. 6, 1095 i.101, 1986). With Xhol and BamHl a DNA fragment of 180 pB in length was cut from the vector pES9.10.9, it was purified by agarose gel electrophoresis and it was cloned in the pA202 vector also cut with Xhol and BamHl and dephosphorylated. By means of this cloning, DesPro2-Arg-15-aprotinin is replaced in the pA202 vector by DesPro2-Serl0-Argl5-AlaI7-As? 24-Thr26-Glu31-Asn41-Glu53-aprotinin. Yeast cells (JC34.4D) were transformed with the vector pES13.10.L resulting from this cloning. Other E. coli / yeast seesaw vectors with different promoters, such as, for example, the constitutive promoter GAPDH or the inducible GAL 10, can be obtained in an analogous manner and also lead to the secretion of DesPro2-Serl0-Argl5-Alal7-Asp24-Thr26 -Glu31-Asn41-Glu53-aprotinin.

Beside this, it is also possible, of course, to use rocker vectors with other origins of replication in yeasts, such as, for example, the chromosomal segment of autonomous replication (ars). Suitable selectable marker genes, together with the URA3 gene, are those genes that contribute to the prototrophy of a yeast auxotrophic mutant, such as, for example, the LEU2, HIS3 or TRP1 genes. In addition, naturally, genes whose products provide resistance to various antibiotics, such as, for example, aminoglycoside G418, can also be used. Other yeasts, such as, for example, the rachetylotrophic yeasts Pichia? Ast_ris or Hansenula polymorpha, after transformation with suitable vectors are likewise able to produce DesPro2-SerlO-Argl5-AlaI7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin. Example 2 Preparation of an expression vector in yeast for the secretion of recombinant Serl0-Arsl5-Alal7-Asp24-Thr26 ~ Glu31-Asn41-Glu53-aprotinin with the native N-terminal sequence "Arq-Pro-Asp". an expression vector in yeast that allows the secretion of Serl0-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin with the native N-terminal sequence "Arg-Pro-Asp", was first amplified by PCR the MFal promoter with the pre-sequence of the factor a and the 5 'end of the aprotinin gene (up to the recognition sequence of the restriction enzyme Xhol) and was cloned. The primers used had the following sequence: Primer C 5'GG \ TATCTATTGATAAGATTTAAAGGTATTTGACAAG 3 '. The recognition sequence of EcoRV is underlined D primer 'GGGCTCGAGGC AGAAATCTGGTCTAGCCAAAGCAGAAGAAGCAGCGAA CAAGACAGCAGTGAAAATAGATGGAATCTCAGGCTTTTAATCGTTTATATT 3 'The Xhol recognition sequence is underlined. The PCR mixture contained 200 ng of plasmid DNA pA202, 0.2 μM of primer C, 0.2 μM of primer D, 200 μM of dNTPs, 1 x PCR reaction buffer 11 (Stratagene, Opti-Prime ") and 2.5 U of Taq DNA polymerase (Perkin Elmer) in a total volume of 50 μl The conditions of the "cycle" were: 1 minute at 942C, 30 cycles respectively with 1 minute at 942C, 1 minute at 50SC and 2 minutes at 72 ° C and a subsequent incubation at 722 ° C for 5 minutes The PCR mixture was diluted 1: 5 and ligated with the pCRII vector (Invítrogen) with E. coli DH5a cells transformed with the ligation mixture. positive were identified after a restriction digestion with the enzyme EcoRI and several clones were sequenced The clone pIU20.11.L was used for the subsequent works.The E. coli / yeast seesaw vector pYES2 (Invitrogen) was used for the construction of a yeast secretion vector, in which the sequence Serl0-Argl5-Alal7-As? 24-Thr26-Glu31-Asn41-Glu53-a Protinin has been directly linked to the pre sequence of the alpha factor of the yeast. First, the vector? YES2 was cut with the restriction enzymes SspI and BamHI, dephosphorylated and gel purified. With this, the GAL1 promoter and the ori fl present on the vector pYES2 are separated. From the pIU20.11.L vector, a DNA fragment about 1030 pB in length was cut with EcoRV and Xhol, purified by agarose gel electrophoresis and cloned, together with an XhoI and BamHI fragment of the vector pES9.10.L of about 180 bp in length, in the pYES2 vector cut with SspI and BamHl. E.coli DH5a cells were transformed with the ligation mixture. Positive clones were identified and sequenced after restriction digestion with the Xhol enzyme. Yeast cells (JC34.4D) were transformed with the vector pIU28.11.L resulting from this cloning. The expression vector pIU28.11.L no longer contains the pro sequence of factor a, so that the processing of Serl0-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-apratinin is effected exclusively through the signal peptidase and is made independently of the dissociation by the KexII protease. EXAMPLE 3 Fermentation of Saccharo vces Cerevisiae An expression strain of Saccharomyces cerevisiae was fermented as described above. Example 4 Purification of aprotinin derivatives without net charge at neutral value of pH 1. Review about suitable purification procedures After fermentation, the cells are separated by centrifugation and the remaining supernatant is filtered to remove residual cells.

The cell-free supernatant is adjusted to pH 3 by the addition of concentrated citric acid. The solution should be diluted conveniently with distilled water, to adjust a conductivity of less than 8 mS / cm. The solution is then applied to a cation exchange column that has previously been equilibrated with an acid buffer. The unbound material is separated by repeated washing with starter buffer. The product is eluted with the aid of a saline gradient. In the obtained fractions, the product content is tested with the help of inverted phase high pressure liquid chromatography (RP-HPLC) and a biological activity assay that determines the inhibition of protease. The fractions containing the product are combined and applied directly to a preparative RP-HPLC column. The column had previously been equilibrated with an acid buffer. The unbound protein is separated by washing the column with starter buffer. The product is eluted with the aid of gradients of an organic solvent. The product content is assayed again in the fractions as described above and those fractions containing product are combined. Depending on the purity reached by the product, it may be necessary to purify the assembled fractions through a second column of RP-HPLC. The conditions are essentially the same as those already described. The product solution obtained is diluted with water for injection and packaged in suitable portions and lyophilized. Other methods for the purification of aprotinin derivatives with no net charge at neutral pH value, which can be combined with the processes described above, are affinity chromatography on trypsin immobilized on sepharose and gel filtration chromatography. 2. Purification of DesPro2-SerlO-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin Material from fermentations of 101 was purified by the following procedure. Once the fermentation had concluded, the contents of the fermenter were adjusted to pH 3 with concentrated citric acid and heated to 70 ° C. for 10 minutes. The cells were then separated by centrifugation (15 minutes, 7,500 x g, Heraeus centrifuge) and the supernatant obtained was filtered (8 μm to 0.2 μm, Millipore, Germany). From this stage the supernatant can be preserved until subsequent use by freezing at -18 ° C. The solution was then diluted by addition of distilled water to a conductivity of less than 8 mS / cm and applied on an SP-Sepharose FF column (Pharmacia, Sweden). The column had previously been equilibrated with 50 nM citrate-NaOH buffer, pH 3. The unbound protein was removed by intensive washing with the same buffer. The product was then eluted with the aid of a saline gradient (CINa ÍM). In the fractions obtained, the product content was tested with the aid of reversed phase high pressure liquid chromatography (RP-HPLC, C4) and by assay of the protease inhibitory activity. Those fractions containing the desired product were pooled. The product solution was then applied directly on the first RP-HPLC column (Source 15 RPC, Pharmacia, Sweden) which had been previously equilibrated with 0.1% trifluoroacetic acid / water. The unbound protein was removed by intensive washing with the same buffer. The product was eluted with the aid of a linear gradient of acetonitrile (0-70%). In the obtained fractions, the product content was again tested with the methods described above and those fractions containing the product were combined. For the subsequent purification, the solution containing the product was diluted with water for injection purposes and applied to a second column of RP-HPLC (Vydac C8, Vydac, USA) which had been previously equilibrated with 0.1% trifluoroacetic acid. /Water. The unbound protein was removed by intensive washing with the same buffer. The product was eluted with the aid of a linear gradient of acetonitrile (0-70%). In the obtained fractions, the product content was again tested with the methods described above and those fractions containing the product were combined.

The solution containing the product was diluted with water for injection purposes, packed in suitable portions (20, 10, 1 and 0.2 mg), lyophilized and analyzed. Example 5 Determination of the Ki value of human plasma kallikrein coBesPro-Serl0-Arsl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin Example: 1 unit of human plasma kallikrein was diluted with buffer to 16 ml (0.05M Tris) / 0.1 M NaCl, 0.05% Tween-20, pH 8.2). 200 μl of this enzyme solution was mixed with decreasing volumes of assay buffer (250, 240, 230, 220, 200, 180, 170, 150, 100, 50 μl) and then increasing amounts of inhibitor were added in the assay buffer (10, 20, 30, 50, 70, 80, 100, 150, 200 and 250 μl, concentration 0.7 μg / μl). The enzyme / inhibitor solution was preincubated at room temperature for 4 hours. Then 180 μl of each solution was added to wells of a microtitre plate and mixed with 20 μl of substrate solution. The change in absorption at 405 nm was measured for 10 minutes. The speed of the enzymatic reactions was determined and from it the Ki value was calculated by the Bieth method (Biochemical Medicine 32: 387-97 (1984)). Substrate stock solution: 0.1M in DMSO Substrate solution: S-2302 1 x 10 M in assay buffer Test buffer: Tris- (hydroxymethyl) -aminomethane 0.05M, CINa 0.1M, 0.05% Tween-20, pH 8.2; 1 ml of benzyl alcohol / 1 The kinetic constants of the complexes with the enzymes plasmin, factor Xla, bovine trypsin and chymotrypsin were determined following the same procedure. Substrates were Chromozym PL for plasmin, HD-Pro-Phe-Arg-pNA for factor XI, S-2444 for trypsin and Suc-Phe-Leu-Phe-pNA for chymotrypsin. Example 6 ~ Results of the protein-protein characterization of DesPro2-Serl0-Argl5-Alal7-Aβp24-Thr2β-Glu31-Asn41-Glu53-aprotinin The protease inhibitor DesPro2-SerlO-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41- Glu53-aprotinin was obtained by secretion by means of a yeast modified by genetic engineering. From the supernatant of the yeast, it was purified by various chromatographic methods, until homogeneous. The following analytical assays of proteins show the identity of the inhibitor with the cloned sequence. Analysis of the N-terminal sequence The protease inhibitor was completely sequenced over 57 stages. The following list shows the determined protein sequence, which is identical to the cloned sequence: i Arg-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Ser-Thr-Gly-Pro-Cys-Arg-Ala-Aia-Ile-Ile-Arg-Tyr- 21 Phe-Tyr-Asp-Ala- Thr-Ala-Gly-Leu-Cys-Glu-Thr-Phe-Val-Tyr-GIy-Gly-Cys-Arg-Ala-40 Asn-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp -Cys-Met-Glu-Thr-Cys-Gly-Gly-Ala Analysis of the sequence of DesPro2-SerlO-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin over 57 steps.

Amino acid analysis The amino acid analysis is an important quantitative parameter for the characterization of a protein. Together with the protein content determines, if the primary structure is known, the number of individual amino acids. The amino acid analysis of DesPro2-Serl0-Argl5-Alal7-As? 24-Thr26-Glu31-Asn41-Glu53-a? Rotinin is in good agreement with the theoretical values of the primary structure (Table 1).

Table 1 Amino acid analysis of DesPro2-SerlO-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin. The integers are referred to Ala = 7. Amino Acid Full number Theoretical numbers CysS03H 5.28 6 Asp 6.27 6 Thr 3.49 4 Ser 1.58 2 Glu 4.25 4 Gly 6.16 6 _ Wing 7.00 7 Val 0.98 1 Met 1.10 1 He 1.66 2 Leu 1.89 2 Tyr 2.49 3 Phe 4.11 4 Lys 1.05 1 Arg 4.73 5 Pro 3.23 3 *) Cysteine and methionine were determined after oxidation with performic acid (Met as methionisulfone).

Chromatography in inverted phase In the HPLC chromatography of proteins chemically linked to inverted phases it is arrived, through a hydrophobic exchange action of the protein, to a union to the phase used. The proteins are expelled, according to the strength of their binding to the stationary phase, by organic solvents (mobile phase). For this reason, this method is a good criterion for estimating the purity of a protein. DesPro2-Seryl-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin elutes from the RP-18 phase in the form of a single peak. It was shown that the isolated protease inhibitor is very pure. CE Chromatography Capillary column electrophoresis allows the separation of peptides and proteins in the electric field due to their charge. In this, the goodness of the separation depends on the buffer, the pH value, the temperature and the additives used. As capillary columns, columns called "fused silica" are used, with an internal diameter of 50-100 μm. DesPro2-SerlO-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin was separated on a "fused silica" column in the electric field. The electrophoretogram shows a fine peak. Molecular weight determination The molecular weight of DesPro2-Seryl-Argl5-Alal7-As? 24-Thr26-Glu31-Asn41-Glu53-a? Rotinin was determined with the MALDI technique in 6.223 Dalton. The molecular weight determined is in good agreement, within the framework of the accuracy of the measurement method, with the theoretical value ofalton. Sinapic acid was used as matrix. Gel electrophoresis with SDS The DesPro2-Serl0-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin was analyzed by electrophoresis with SDS under reducing and non-reducing conditions. It shows a band in the range of about 6.5 kD. Example 7 Determination of Ki value for enzyme complexes with DesPro2-Serl0-Arql5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin The inhibitory constants of DesPro2-Seryl-Argl5-Alal7-Asp24-Thr26- were determined Glu31-Asn41-Glu53- aprotinin for various enzymes. Table 2 reproduces the Ki values. Table 2: Inhibitory constants of the complexes of DesPro2-SerlO-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin with the enzymes calicrein of plasma, factor Xla, bovine uiraotrypsin and bovine trypsin.

EXAMPLE 8 Exchange Activity of Protease Inhibitors with Rabbit Anti-aprotinin Polyclonal or Human Antibodies The cross-reactivity of protease inhibitors obtained recombinantly with rabbit or human polyclonal anti-aprotinin antibodies was tested. It was found that the various variants of protease inhibitors show only very weak exchange activity with aprotinin antisera.

SECUENCIATION PROTOCOL (1) GENERAL INFORMATION: (i) APPLICANT (A) NAME: Bayer AG (B) STREET: Bayerwerk (C) PLACE: Leverkusen (E) COUNTRY: Germany (F) POSTAL SECTION: 51368 (G) TELEPHONE: 0214/3061455 (H) TELEFAX: 0214/303482 (J) DESIGNATION OF THE INVENTION: Aprotinin variants with improved properties, (iii) NUMBER OF SEQUENCES: 5 (IV) LEGIBLE FORM BY COMPUTER: (A) DATA SUPPORT: Disquette . (B) COMPUTER: PC-IBM, compatible. (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Relay # 1.0, Version 1.30B (EPA). (2) DATA OF SEQUENCE ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 69 base pairs. (B) TYPE: Nucleotide. (C) SHAPE OF THE HEBRA: Single chain. (D) TOPOLOGY: Linear. (ii) CLASS OF MOLECULE: genomic DNA. (iii) HYPOTHETICAL: none, (iv) ANTIPARALELO: none, (vi) ORIGINAL PROVENANCE: (A) ORGANISM: primer A (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO. 1: GGCTGCAGAG CTAACCGTAA CAACTTCAAA TCCGCGGAAG ACTGCATGGA AACTTGCGGT 60 GGTGCTTAG_69_(2) SEQUENCE INFORMATION ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 93 base pairs. (B) TYPE: Nucleotide. (C) SHAPE OF THE HEBRA: Single chain. (D) TOPOLOGY: Linear. (ii) CLASS OF MOLECULE: genomic DNA. (ii) HYPOTHETICAL: none, (iv) ANTIPARALELO: none, (vi) ORIGINAL PROVENANCE: (A) ORGANISM: primer B (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2: TGCCTCGAGC CGCCGTCTAC TGGGCCCTOC AGAGCTATCA TCCGTTACTT CTACGATGCA 60 ACTGCAGGCC TGTGTGAAAC CTTCGTATAC GGC 93 (2) SEQUENCE INFORMATION ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs. (B) TYPE: Nucleotide. (C) SHAPE OF THE HEBRA: Single chain.

(D) TOPOLOGY: Linear, (ii) CLASS OF MOLECULES: Genomic DNA. (v) FRAGMENT CLASS: linear, (vi) ORIGINAL ORIGINAL: (A) ORGANISM: primer C (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: GGGATATCTA TTGATAAGAT TTAAAGGTAT TTGACAAG (2) SEQUENCE INFORMATION ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 99 base pairs. (B) TYPE: Nucleotide. (C) SHAPE OF THE HEBRA: Single chain. (D) TOPOLOGY: Linear. (ii) CLASS OF MOLECULE: genomic DNA. (ii) HYPOTHETICAL: none, (iv) ANTIPARALELO: none, (vi) ORIGINAL PROVENANCE: (A) ORGANISM: primer D (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 4: GGGCTCGAGG CAGAAATCTG GTCTAGCCAA AGCAGAAGAA GCAGCGAACA AGACAGCAGT 6C GAAAATAGAT GGAATCTCAT TCTTTTAATC GTTTATATT ge (2) SEQUENCE INFORMATION ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 57 amino acids. (B) TYPE: Amino acid. (C) SHAPE OF THE HEBRA: Single chain.

(D) TOPOLOGY: Linear, (ii) CLASS OF MOLECULE: Peptide. (v) FRAGMENT CLASS: linear, (vi) ORIGINAL ORIGINAL: (A) ORGANISM: Aprotinin variant (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 5: Arg Asp Phe Cys Leu Glu Pro Pro Be Thr Gly Pro Cys Arg Wing Ala 1 5 10 15 lie He Arg Tyr Phe Tyr Asp Ala Thr Ala Gly Leu Cys Glu Thr Phe u 25 25 Val Tyr Gly Gly Cys Arg Ala Asn Arg Asn Asn Phe Lys "be Ala Glu 35 40 5 A = p Cvs Met Glu Thr Cys Gly Gly Ala It is noted that, in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (5)

1. CLAIMS 1. Aprotinin variants with a net charge of +3 to -3 at pH 7 and with the amino acids Argl5 or Argl5-Alal7 in the binding region.
2. 2. Aprotinin variants according to claim 1 for the inhibition of serine proteases.
3. 3. Aprotinin variants according to claim 1 or 2 with a modified N-terminal sequence.
4. 4. Aprotinin variants according to claim 1 or 2 with an N-terminal elongation or shortening or with amino acids deleted in the N-terrnin position. 5.- Aprotinin variant of the group DesPro2-SerlO-Argl5-Alal7-Asp24- Thr26-Glu31-Asn41-Glu53-aprotinin, DesPro2-Serl0-Argl5-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin, DesPro2-Serl0-Argl5-Serl7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin, DesPro2- SerlO-Argl5-Alal7-Thr26-Glu31-Asn41-Glu53-aprotinin, DesPro2-Serl0-Argl5-Alal7-Asp24-Thr26-Asn41-Glu53-aprotinin, Serl0-Argl5-Alal7-Asp24-Thr26-Glu31-Asn41-Glu53- aprotinin, Serl0-Argl5-Asp24-Thr26-Glu31-Asn41- Glu53-a? rotinin, Serl0-Argl5-Serl7-Asp24-Thr26-Glu31-Asn41-Glu53-aprotinin, Serl0-Argl5-Alal7-Thr26-Glu31-Asn41- Glu53-aprotinin and Serl0-Argl5-Alal7-Asp24-Thr26-Asn41-Glu53-aprotinin. 6. Medicaments, characterized in that they contain one or more of the aprotinin variants according to claims 1 to
5. 5. Use of aprotinin variants according to claims 1 to 5 for the production of drugs for application in surgery, to reduce the loss of blood in polytrauma, shock and inflammation.