Patent application title: Proteolysis Resistant Active VEGF
Sabine Eming (Cologne, DE)
Thomas Krieg (Cologne, DE)
Stephan Sollberg (Schwerin, DE)
Gereon Lauer (Radolfzell, DE)
bayer Innovation GmbH
IPC8 Class: AA61K3818FI
Publication date: 2012-10-11
Patent application number: 20120258915
The invention relates to endothelial growth factor (VEGF) in which the
alanine at AA position 111 is replaced by proline. The arginine at AA
position 110 may moreover be replaced by another amino acid. The
invention also relates to derivatives of the VEGF according to the
invention, nucleic acids, expression systems, medicaments and the use of
the VEGF mutants of the invention for the treatment of chronic wounds.
17. A method for inhibiting plasmin, inducing neoangiogenesis, and/or inhibiting matrix degradation comprising administering to a patient in need thereof an effective amount of a VEGF variant comprising: an amino acid sequence, wherein said amino acid sequence is the amino acid sequence of a native vascular endothelial growth factor in which at least one amino acid at positions 110 or 111 of the native vascular endothelial growth factor is replaced by proline or is deleted, wherein plasmin is inhibited, neoangiogenesis is induced and/or matrix degradation is inhibited.
18. The methods of claim 17, wherein at least one further amino acid at the other of the positions 110 or 111 of the VEGF variant is replaced or is deleted.
19. The methods of claim 17, wherein an alanine at position 111 of the native vascular endothelial growth factor is replaced by proline.
20. The methods of claim 19, wherein an arginine at position 110 of the native vascular endothelial growth factor is replaced by another amino acid.
21. The methods of claim 20, wherein said another amino acid is proline.
22. The methods of claim 17, wherein an arginine at position 110 of the native vascular endothelial growth factor is replaced by proline.
23. The methods of claim 17, wherein an alanine at position 111 of the native vascular endothelial growth factor is replaced by another amino acid.
24. The method of claim 23, wherein said another amino acid is proline.
25. The methods of claim 17, wherein the VEGF variant is in the form of any of the splice variants VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 or VEGF.sub.206.
26. The methods of claim 17, wherein the VEGF165 variant has one of the amino acid sequences SEQ ID NO:1 or SEQ ID NO:2.
27. The methods of claim 17, wherein the amino acid chain is modified or derivatized and/or comprises mutations, insertions and/or deletions and/or has a signal sequence.
28. The method of claim 27, wherein the signal sequence is connected N-terminally to the amino acid chain of the VEGF variant and has the sequence of SEQ ID NO:3.
29. The method of claim 17, wherein the an amino acid at position 111 of the native vascular endothelial growth factor is replaced by proline or is deleted and wherein an amino acid at position 110 is maintained or replaced with lysine.
30. The method of claim 29, wherein the amino acid at position 111 of the native vascular endothelial growth factor is replaced by proline.
31. The method of claim 30, wherein the amino acid at position 110 of the native vascular endothelial growth factor is replaced by lysine.
CROSS-REFERENCE TO RELATED APPLICATIONS
 This is a divisional application of U.S. application Ser. No. 12/349,531, filed Jan. 7, 2009 which is a divisional application of U.S. application Ser. No. 10/506,893, filed Sep. 1, 2005, which is incorporated herein by reference in its entirety and which is the U.S. National Stage of International Application No. PCT/EP2003/02289, filed Mar. 6, 2003 designating the United States and claiming priority to European Application No. 02005186.8, filed Mar. 8, 2002.
FIELD OF THE INVENTION
 The invention relates to vascular endothelial growth factor (VEGF) in which the alanine at AA position 111 is replaced by proline. The arginine at AA position 110 may moreover be replaced by another amino acid. The invention also relates to derivatives of the VEGF according to the invention, nucleic acids, expression systems, medicaments and the use of the VEGF mutants of the invention for the treatment of chronic wounds.
BACKGROUND OF THE INVENTION
 An important stage in cutaneous wound healing is the formation of a granulation tissue. Firstly associated with the latter is the migration in of newly formed vessels (neoangiogenesis). Numerous experimental and clinical studies show that chronic wounds are characterized by impaired angiogenesis and thus diminished formation of granulation tissue.
 A large number of mediators which stimulate angiogenesis during wound healing are known. They include firstly the factors which, besides stimulating endothelial cells, also activate mesenchymal and/or epidermal cells (bFGF, aFGF, TGF-a, PDGF), and secondly so-called endothelial cell-specific factors whose receptors are substantially confined to endothelial cells (VEGF, angiopoietin). A large number of physiological and pathological reactions involving the blood vessels correlates with an increased expression of VEGF and its receptors, so that VEGF assumes a central role in angiogenesis of the skin. The first indications of the possible importance of VEGF in wound healing impairments were provided on the basis of experiments on VEGF expression in diabetic mice (db/db mice) (Frank et al. 1995). It was possible to show in this model that the wound healing impairment correlates with a diminished VEGF expression. It has recently been possible to provide support for the role of VEGF in wound healing by a further transgenic animal model (Fukumura et al., 1998) and detection of VEGF in the wound discharge from acute human wounds (Nissen et al., 1998).
 It has further been shown that there is increased expression of the mRNA of VEGF and its receptors in the tissue of chronic wounds (Lauer et al., 2000). Investigations by SDS-PAGE show, however, breakdown of the VEGF protein in the chronic wound environment, in contrast to the acute wound. This breakdown leads to a significant loss of the biological activity and may thus, despite the increased expression of the VEGF receptors, underlie a deficient stimulation of neoangiogenesis in the chronic wound environment. As explained above, it was possible to show that plasmin is involved in the cleavage of VEGF in the chronic wound environment (Lauer et al., 2000).
 Cleavage of VEGF165 via plasmin leads to detachment of the carboxyl-terminal domain which is encoded by Exon 7. Whereas Exons 3 and 4 determine the binding properties of VEGF to the VEGF receptors Flt-1 and Flk-1/KDR, Exon 7 has a critical importance in the interaction of VEGF with neuropilin-1 (Keyt et al. 1996). Neuropilin-1 is a 130 kDa cell surface glycoprotein. Its role in the potentiation of the mitogenic effect of VEGF on endothelial cells was described only recently (Soker et al. 1998). In this connection, the interaction of neuropilin-1 with Flk-1/KDR appears to be important because binding solely of VEGF to neuropilin-1 has no signal effect.
 Plasmin belongs to the class of serine proteases. These enzymes are able to cleave peptide linkages. The cleavage takes place by a so-called catalytic triad. In the catalytic centre thereof an essential part is played in particular by the eponymous serine, but also by the amino acids histidine and aspartate, because the process of peptide cleavage takes place by means of them (Stryer 1987, pp. 231 et seq.). Although the mechanism of the linkage cleavage is identical in all serine proteases, they differ markedly in their substrate specificity. Thus, plasmin, just like trypsin, cleaves peptide linkages after the basic amino acids lysine and arginine. However, the substrate specificity of plasmin, which is determined by the structure of the catalytic centre, leads to plasmin being unable to cleave all these linkages. Catalysis of peptide-linkage cleavage is possible only if the corresponding protein segments are able to interact with the catalytic centre of the enzyme (Powers et al. 1993; Stryer 1987). To date, no unambiguous consensus sequence of a plasmin cleavage site is known.
 The present invention is based on the object of providing improved means for healing chronic wounds. Surprisingly, this object is achieved by the provision according to the invention of a vascular endothelial growth factor (VEGF) variant which is characterized in that at least one amino acid in the sequence of the native vascular endothelial growth factor at positions 109 to 112 of the native vascular endothelial growth factor is replaced by another amino acid or a deletion.
BRIEF SUMMARY OF THE INVENTION
 In one embodiment of the invention, at least one amino acid in the sequence of the native vascular endothelial growth factor is replaced by proline in the VEGF variant according to the invention the positions 109 to 112. In a further embodiment, besides proline, at least one further amino acid at one of positions 109 to 112 in the VEGF according to the invention is replaced or a deletion.
 In a further embodiment, the alanine at AA position 111 of the native vascular endothelial growth factor is replaced by proline in the VEGF variant according to the invention.
 In another embodiment, the arginine at AA position 110 of the native vascular endothelial growth factor is replaced by another amino acid in the VEGF variant according to the invention. In particular, the arginine at AA position 110 of the native vascular endothelial growth factor is replaced by proline.
 In a further embodiment of the invention, the alanine at AA position 111 of the native vascular endothelial growth factor is replaced by another amino acid in the VEGF.
 It is possible in particular for the arginine at AA position 110 of the native vascular endothelial growth factor and the alanine at AA position 111 of the native vascular endothelial growth factor to be replaced by proline in the VEGF variant according to the invention.
 The VEGF mutants according to the invention are preferably in the form of one of the splice variants VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 or VEGF206.
 The VEGF mutants according to the invention display not only markedly increased stability towards plasmin, but also an activity comparable to that of wild-type VEGF. Surprisingly, the VEGF variants according to the invention additionally display distinctly increased stability in chronic wound fluids.
 The mutations have been carried out at a site which is critical for the biological activity of the VEGF molecule. There was thus a fear that a change in the protein structure in this region has a negative effect on the activity of VEGF165. The amino acid proline, which is introduced according to the invention at position 111, is a cyclic a-imino acid. Owing to the cyclic form of the pyrrolidine residue, it has a rigid conformation which also has an effect on the structure of the respective proteins. Thus, proline acts for example as a strong a-helix breaker. It is therefore particularly surprising that replacement precisely of alanine at position 111 by proline generates a VEGF mutant which is stable towards the protease plasmin, is stable in chronic would fluids and, at the same time, still has an activity corresponding to that of the wild-type protein.
 The invention relates in particular to VEGF variants of the two sequences SEQ ID NO: 1 or SEQ ID NO: 2.
 The invention also relates to variants of the VEGF mutants mentioned above, in which the amino acid sequences are modified or derivatized, or comprise mutations, insertions or deletions. This relates in particular to VEGF variants in which further single amino acids are replaced, and those which are glycosylated, amidated, acetylated, sulphated or phosphorylated. Such VEGF variants preferably have an activity comparable to or higher than the wild-type VEGF.
 The VEGF variants according to the invention may also have a signal sequence. The signal sequence may be connected N-terminally to the amino acid chain of the VEGF variant and have the sequence
TABLE-US-00001 (SEQ ID NO: 3) Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gln Ala.
 The invention also relates to nucleic acids which code for the abovementioned VGEF mutants, and vectors for VEGF expression which comprise such nucleic acids.
 The invention relates to a medicament which comprises the abovementioned mutants of VEGF, and to the use of the VEGF mutants for producing a medicament for the treatment of chronic wounds, caused by vascular lesions such as chronic venous insufficiency (CVI), primary/secondary lymphoedema, arterial occlusive disease, metabolic disorders such as diabetes mellitus, gout or decubitus ulcer, chronic inflammatory disorders such as pyoderma gangrenosum, vasculitis, perforating dermatoses such as diabetic necrobiosis lipoidica and granuloma annulare, haematological primary disorders such as coagulation defects, sickle cell anaemia and polycythemia vera, tumours, such as primary cutaneous tumours and ulcerative metastases, and for plasmin inhibition, for inducing neoangiogenesis and/or for inhibiting matrix degradation.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1, consisting of FIGS. 1A and 1B, shows that the VEGF165 mutations are resistant to cleavage by plasmin. The figure shows incubation of VEGF165 and the mutated proteins in a plasmin solution [0.01 U/ml] or buffer solution (FIG. 1B, lanes 18, 19) for the stated periods. Analysis of the degradation behavior took place by Western blotting and immunodetection.
 FIGS. 2A to 2C show that the Ala111 to Pro111 mutation increases the stability of VEGF in chronic wound fluid. FIG. 2A: VEGF165 wild type expressed in COS-1 cells. FIG. 2B: the VEGF variants were incubated in chronic wound fluid for the stated periods, and the degradation behavior was visualized by immunodetection. In this case, wound fluids from two different patients were investigated: patient X, lanes 1-16; patient Y: lanes 17-20). FIG. 2C: Densitometric visualization of the degradation of VEGF wild type and Mut.sub.Lys-Pro in chronic wound fluid. The relative signal strength from three independently performed Western blot analyses (mean+/-SD) is shown.
 FIG. 3 shows that the VEGF mutants are biologically active. VEGF165 wild type and VEGF mutants were each incubated in increasing concentrations with HUVE cells. The rate of incorporation of the base analogue into the DNA of the proliferating cells determined by BrdU ELISA is shown (mean+/-SD; n=3).
 FIGS. 4A to 4D show that plasmin does not alter the biological activity of the VEGF165 mutants. A comparison is shown of the relative BrdU incorporation into HUVE cells through stimulation with VEGF165 wild type (FIG. 4A, FIG. 4B), MutAla (FIG. 4C) and Mut.sub.Lys-Pro (FIG. 4D) after incubation of the stated protein in buffer or plasmin (means+/-SD; n=3).
DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS OF THE INVENTION
 Topical use of growth factors represents a novel therapeutic concept in wound healing. It has been possible to observe an improvement in the healing of chronic wounds in a large number of clinical studies with the use of EGF, bFGF, PDWHF and PDGF (Scharffetter-Kochanek et al. 2000). However, a criticism which should be noted is that the results of these studies did not come up to the expectations which existed in view of the good activity of these mediators in animal models (Lawrence et al. 1994). This restricted activity of the growth factors is certainly substantially explained by the increased proteolytic activity in the chronic wound environment, which leads to degradation of the topically applied factors. It is thus clear that local wound management by administration of growth factors represents a promising novel therapeutic strategy. However, it is necessary to develop strategies which control the proteolytic activity in the chronic wound environment. The production of master cytokines with increased stability in the chronic wound environment certainly represents a novel therapeutic approach in this connection. The VEGF mutants according to the invention are particularly suitable, because of their high stability in the wound fluid, for the topical treatment of chronic wounds.
 Four mutants were produced by site-directed mutagenesis by carrying out targeted amino acid replacements at Arg110 and Ala111. The cDNA which codes for human VEGF165 was cloned into the SV40 replication expression vector pcDNA 3.1 (from Invitrogen, De Schelp, NL) using the BamHI and EcoRI cleavage sites in the cloning site. The Gene Editor® system from Promega (Mannheim) was used for the site-directed in vitro mutagenesis. This system is based on annealing of oligonucleotides which harbour the appropriate mutation onto the initial sequence. The initial sequence of VEGF165 in the region of the mutations is:
TABLE-US-00002 106 107 108 109 110 111 112 113 (SEQ ID NO: 4) GA CCA AAG AAA GAT AGA GCA AGA CAA G (SEQ ID NO: 5) Pro Lys Lys Asp Arg Ala Arg Gln
 To introduce the mutations, the following mismatch oligonucleotides were used as primers:
TABLE-US-00003 Mutation 1: MutAla: (SEQ ID NO: 6) GA CCA AAG AAA GAT GCC GCA AGA CAA G (SEQ ID NO: 7) Pro Lys Lys Asp Ala Ala Arg Gln Mutation 2: MutGln: (SEQ ID NO: 8) GA CCA AAG AAA GAT CAG GCA AGA CAA G (SEQ ID NO: 9) Pro Lys Lys Asp Gln Ala Arg Gln Mutation 3: MutPro: (SEQ ID NO: 10) GA CCA AAG AAA GAT AGG CCA AGA CAA G (SEQ ID NO: 11) Pro Lys Lys Asp Arg Pro Arg Gln Mutation 4: Mut.sub.Lys-Pro: (SEQ ID NO: 12) GA CCA AAG AAA GAT AAG CCA AGA CAA G (SEQ ID NO: 13) Pro Lys Lys Asp Lys Pro Arg Gln
 The mutagenesis primers used are each detailed with the modified amino acid sequences obtained therewith. The regions with the bases or amino acids which are changed from the wild-type sequence are in italics.
 In mutation 1, arginine110 was replaced by a nonpolar alanine. In mutation 2, a polar, uncharged glutamine was introduced at the same position. In mutant 3, the alanine at position 111, not the basic arginine110, was replaced by a proline. In mutant 4, two amino acids were replaced. In this case, lysine and proline were introduced in place of arginine110 and alanine111. After the mutagenesis had been carried out, the mutations were verified by sequence analysis. The resulting VEGF mutants had the following sequences for amino acids 109-112:
TABLE-US-00004 VEGF165 wild type: (SEQ ID NO: 14) -Asp109Arg110Ala111Arg112- MutGln: (SEQ ID NO: 15) -Asp109Gln110Ala111Arg112- MutAla: (SEQ ID NO: 16) -Asp109Ala110Ala111Arg112- MutPro: (SEQ ID NO: 17) -Asp109Arg110Pro111Arg112- Mut.sub.Lys-Pro: (SEQ ID NO: 18) -Asp109Lys110Pro111Arg112-
 The mutants MutPro and Mut.sub.Lys-Pro are mutants according to the invention, whereas MutGln and MutAla are produced and investigated for the purposes of comparison. The resulting VEGF165 expression vectors were used in the further investigations.
Production of Recombinant VEGF165 Protein
 VEGF165 protein was expressed in eukaryotic COS-1 cells. The pcDNA 3.1 expression vector used comprises an SV-40 origin of replication. This serves to amplify the vector in cells which express a large T antigen of the SV-40 virus. The COS-1 cells used possess a corresponding element integrated into the genome, so that episomal replication of the vector results. Expression of the target protein VEGF for several days is achieved thereby without stable integration (transformation) of the vector into the cell genome. The COS-1 cells were transfected with the expression plasmids obtained in the mutagenesis. For this purpose, the Superfect transfection reagent (QIAGEN, Hilden) was used according to the manufacturer's protocols.
 Like a large number of growth factors, VEGF165 also has a heparin-binding site which is located at the basic C terminus. The binding to heparin was exploited for purification of the protein by affinity chromatography (Mohanraj et al. 1995). The VEGF and VEGF variants were isolated by the following steps:
 The COS-1 cells transformed with the expression plasmids were cultivated in serum-free DMEM (Dulbecco's modified Eagle's medium) comprising 10% fetal calf serum (FSC), 2 mM L-glutamine, penicillin (10 U/ml) and streptomycin (10 μg/ml) and ITS supplement (Sigma, Deisenhofen). Conditioned medium (200 ml) was collected after 48 h and incubated with 5 ml of heparin-Sepharose (Pharmacia, Freiburg) at 4° C. for 4 hours. The heparin-Sepharose was packed into a column. The latter was loaded with culture medium at a flow rate of 25 ml/h. The following steps were carried out:
A: Affinity chromatography with heparin-Sepharose  1. Washing: 0.1 M NaCl; 20 mM Tris/pH 7.2  2. Washing: 0.3 M NaCl; 20 mM Tris/pH 7.2  3. Elution: 0.9 M NaCl; 20 mM Tris/pH 7.2 B: Analysis of the resulting fractions by Western blot analysis C: Desalting of the VEGF-containing fractions by gel filtration  Running buffer: 10 mM Tris/pH 7.2 D: Lyophilization of the solution and determination of the concentration by ELISA
 The resulting VEGF was investigated by SDS-PAGE. The VEGF protein obtained from COS-1 cells differs in its migration behaviour in SDS-PAGE from the commercially available VEGF165 protein used (from R&D Systems). In addition to the signal to be detected at 42 kDA (FIG. 1, lane 6), a further band with a molecular weight which is a few kDA higher is also evident. The reason for this double band of the VGEF protein expressed in COS-1 cells is an altered glycosylation of the growth factor. On expression of VEGF in COS-1 cells there is formation of two differently glycosylated proteins. One form (42 kDa) is identical in its glycosylation to the recombinant VEGF165 which has been used to date and which was produced in insect cells using a baculovirus expression system (R&D Systems, FIG. 1, lane 1). It has an N-glycosylation on the amino acid asparagine at position 74 (Gospodarowicz et al. 1989; Keck et al. 1989). The second band at a higher molecular weight (45 kDa) results from further glycosylation of the protein. The difference in the glycosylation is known for expression in COS cells and has no effect on the biological activity of the growth factor (R&D Systems).
Characterization of the Biochemical and Biological Properties of the Purified VEGF165 Proteins
I. Analysis of the Stability of the VEGF165 Proteins and its Mutations:
a) Incubation in Plasmin
 The four purified mutated VEGF proteins were initially investigated for their stability towards the protease plasmin It was investigated whether the mutations carried out lead to an altered degradation behaviour compared with wild-type VEGF.
 FIG. 1 shows the results of incubation of the VEGF wild type and the VEGF mutants with plasmin Incubation of the VEGF wild type synthesized in COS-1 cells (A, lane 6-10) shows degradation of the growth factor after only 15 minutes. In this case, accurate determination of the size of the resulting fragments by SDS-PAGE is difficult because the signals overlap with the two bands of the differently glycosylated protein. However, the degradation pattern is similar to that of the commercially obtainable VEGF165 (FIG. 1A, lane 1-5). Thus, a fragment with a molecular weight of 38 kDA can be detected after 45 minutes. This corresponds to the 110 dimer fragment of the less glycosylated VEGF variant. These results clearly show that the VEGF protein expressed in the COS-1 cells is also cleaved by plasmin under the chosen conditions.
 FIG. 1B (lane 1-17) shows the results of incubation of mutated proteins. Incubation of the arginine to alanine mutation is shown first (lane 1-5). At zero incubation time, two bands are detectable for the differently glycosylated variants of the VEGF protein, as with the wild type. However, in this case, because of the higher signal intensity, they cannot be differentiated from one another so clearly as with the VEGF165 wild type. In contrast to the VEGF wild type, the mutated protein shows no change in the migration behaviour up to 240 minutes after incubation.
 This observation suggests that the arginine110 to alanine110 mutation has led to inactivation of the plasmin cleavage site. As shown further in FIG. 1B, the three other mutants MutPro, MutGln and Mut.sub.Lys-Pro also show a comparable stability of the signal bands at 45 and 42 kDA after incubation with plasmid for 240 minutes. A control in which the VEGF165 wild type was incubated with plasmin buffer at 37° C. for 4 hours is not degraded (lanes 18 and 19). Overall, these experiments indicate that the produced and purified VEGF mutants are stable towards the protease plasmin.
b) Incubation in Acute and Chronic Wound Fluid
 In the next step, the degradation of the VEGF mutants in wound fluid from patients with acute and chronic wounds was analysed. On incubation of the VEGF165 wild type and all VEGF mutants in acute wound fluid, no degradation was detectable after 240 minutes.
 FIG. 2 shows the effect of chronic wound fluid on the stability of the VEGF proteins. Incubation of the VEGF wild type synthesized in COS-1 cells (FIG. 2A, lane 1-4) for 240 minutes shows degradation of the growth factor with a fragment of about 38 kDa. This corresponds to the 110 dimer fragment of the less glycosylated VEGF variant.
 In contrast to the wild type, the VEGF165 mutants show a different degradation behaviour on incubation in chronic wound fluid. On the one hand, the degradation process observed in the mutations MutGln (FIG. 2B, lanes 13-16 and MutAla (lanes 5-8) is comparable to that of the wild type. Fragments with a molecular weight of about 38 kDa are produced after only about 20 min.
 On the other hand, analysis of the mutants MutPro (lanes 9-12) and Mut.sub.Lys-Pro (Lanes 1-4, 17-20) shows a breakdown behaviour different from the wild type and the mutants MutAla and MutGln. A stable signal at 42 and 45 kDa is seen in the SDS-PAGE up to 60 minutes after incubation. This indicates stabilization of the mutated proteins MutPro and Mut.sub.Lys-Pro in the chronic wound fluid. This difference in the degradation behaviour of the mutants with neutral/nonpolar amino acid and those with proline suggests that further proteases, besides plasmin, are involved in the breakdown of VEGF in the chronic wound environment.
 Degradation is observable with all mutated proteins 240 minutes after incubation in chronic wound fluid. In these cases there is not just formation of clearly defined breakdown fragments; on the contrary, a diffuse signal between 38 and 45 kDa appears after 240 min. This presumably involves proteolysis in the region of the first 20 amino acids (recognition site of the antibody), because the signal strength decreases markedly after 240 min.
 In summary, the results indicate that the VEGF mutants with proline at position 111 are initially stabilized in chronic wound fluid but are degraded in the long term. Comparable results were observed in the would fluids from three different patients with chronic venous insufficiency. The experiments for the various wound fluids were repeated at least twice (FIG. 2B: patient X lanes 1-4; patient Y lanes 17-20). The resulting band pattern always remained the same moreover.
 FIG. 2C shows a densitometric evaluation of the breakdown of VEGF wild type and Mut.sub.Lys-Pro. The aim of the investigation was to quantify the stabilization of the VEGF mutant in the chronic wound fluid. For this purpose, the time-dependent change in the signal strength at the level of the initial signal (42-45 kDa region) compared with the signal at time zero was determined. The densitometric densities measured at the various times are depicted as percentage of the initial signal. It is clear in this densitometric investigation that at every measurement time the VEGF mutant shows a stronger signal by comparison with the VEGF wild type in the 42-45 kDa region, and thus intact VEGF165 protein is present. This observation suggests that this mutation leads to an improved stability and bioactivity of the VEGF protein in the chronic wound environment. The difference between wild type and mutant is statistically significant only 20 minutes after the incubation. The measurements were carried out with identical wound fluid for three independent experiments.
II. Investigations of the Biological Activity of VEGF165 Wild Type and the Mutated Variants:
 It was investigated whether the mutations have an effect on the biological activity of the VEGF molecule. The biological activity was assayed by means of a BrdU proliferation assay (Roche Diagnostics, Mannheim) on human umbilical vein endothelial cells (HUVE cells) in accordance with the manufacturer's information. This entailed the HUVE cells being cultivated with addition of various VEGF mutants, then incubated with BrdU solution for 6 hours and fixed, after which an ELISA was carried out using a BrdU-specific antibody.
 VEGF concentrations between 1 ng/ml and 25 ng/ml were employed. Commercially available recombinant VEGF165 protein (R&D Systems) and VEGF165 wild type synthesized in COS-1 cells showed a half-maximum stimulation of BrdU incorporation at about 3 ng/ml (FIG. 3). The mutated VEGF proteins are characterized by a stimulation of endothelial cell proliferation which is comparable to the VEGF wild type synthesized in COS-1 cells. The maximum stimulation of all the proteins synthesized in COS-1 cells was less than that by commercially obtainable VEGF165 wild type. The reason for the difference between the two curved profiles may be the different expression systems and purification methods for the proteins (Mohanraj et al. 1995). The biological activity of VEGF165 is thus not significantly affected by the mutations carried out.
 The question of the extent to which the biological activity of the VEGF165 wild type and of the VEGF mutants is affected after plasmin incubation was subsequently examined. For this purpose, the VEGF proteins were incubated with plasmin, and then the biological activity was investigated by means of a BrdU proliferation assay on HUVE cells.
 In the graphical representation (FIG. 4), the BrdU incorporation is shown as percentage of the initial signal (time t=0). Incubation of the VEGF wild types (synthesized in COS-1 cells and from R&D Systems) and of the VEGF mutants MutAla and Mut.sub.Lys-Pro in plasmin buffer at 37° C. shows no impairment of the biological activity of the proteins (FIG. 4A-D). In contrast thereto, incubation of the VEGF165 wild types in plasmin leads to a marked reduction in the biological activity (FIG. 4A, B). An activity loss of at least 20% is seen only 20 minutes after incubation, and then falls further to about 50% of the initial activity after 240 minutes. The MutAla and Mut.sub.Lys-Pro mutants show no significant activity loss after incubation with plasmin (FIG. 4C, D). These results underline the "plasmin resistance" of the mutants demonstrated in the Western blot (FIG. 1) and show that the mutated proteins are stable even after incubation with plasmin.
 The introduced mutations thus result in an inhibition of VEGF cleavage by plasmin. A stabilization of VEGF and thus an increased biological activity in the chronic wound environment can be brought about by the Ala111 to Pro111 mutation.
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181165PRTArtificialmutated human VEGF 1Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys1 5 10 15Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25 30Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 40 45Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu 50 55 60Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile65 70 75 80Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Pro Arg 100 105 110Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe 115 120 125Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser 130 135 140Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys145 150 155 160Asp Lys Pro Arg Arg 1652165PRTArtificialmutated human VEGF 2Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys1 5 10 15Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu 20 25 30Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys 35 40 45Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu 50 55 60Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile65 70 75 80Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe 85 90 95Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Lys Pro Arg 100 105 110Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe 115 120 125Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser 130 135 140Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys145 150 155 160Asp Lys Pro Arg Arg 165326PRTHomo sapiens 3Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu1 5 10 15Tyr Leu His His Ala Lys Trp Ser Gln Ala 20 25427DNAHomo sapiens 4gaccaaagaa agatagagca agacaag 2758PRTHomo sapiens 5Pro Lys Lys Asp Arg Ala Arg Gln1 5627DNAArtificialmutated human VEGF sequence 6gaccaaagaa agatgccgca agacaag 2778PRTArtificialmutated human VEGF fragment 7Pro Lys Lys Asp Ala Ala Arg Gln1 5827DNAArtificialmutated human VEGF sequence 8gaccaaagaa agatcaggca agacaag 2798PRTArtificialmutated human VEGF fragment 9Pro Lys Lys Asp Gln Ala Arg Gln1 51027DNAArtificialmutated human VEGF sequence 10gaccaaagaa agataggcca agacaag 27118PRTArtificialmutated human VEGF fragment 11Pro Lys Lys Asp Arg Pro Arg Gln1 51227DNAArtificialmutated human VEGF sequence 12gaccaaagaa agataagcca agacaag 27138PRTArtificialmutated human VEGF fragment 13Pro Lys Lys Asp Lys Pro Arg Gln1 5144PRTHomo sapiens 14Asp Arg Ala Arg1154PRTArtificialmutated human VEGF fragment 15Asp Gln Ala Arg1164PRTArtificialmutated human VEGF fragment 16Asp Ala Ala Arg1174PRTArtificialmutated human VEGF fragment 17Asp Arg Pro Arg1184PRTArtificialmutated human VEGF fragment 18Asp Lys Pro Arg1
Patent applications by Gereon Lauer, Radolfzell DE
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