WO1999050290A9

WO1999050290A9 - Para-poxvirus-coded vascular endothelial cell growth factor (ppv-vegf)

Info

Publication number: WO1999050290A9
Application number: PCT/EP1999/002057
Authority: WO
Inventors: Christoph Dehio; Marlene Roettgen; Hanns-Joachim Rziha; Mathias Buettner
Original assignee: Max Planck Gesellschaft; Christoph Dehio; Marlene Roettgen; Rziha Hanns Joachim; Mathias Buettner
Priority date: 1998-03-27
Filing date: 1999-03-26
Publication date: 1999-12-09
Also published as: DE19813774A1; AU3147799A; WO1999050290A1

Abstract

The invention relates to novel polypeptides which can be obtained from para-poxviruses and which have homologies with human VEGF proteins and are able to stimulate the proliferation of endothelial cells. These VEGF-PPV polypeptides are suitable for use in pharmaceutical applications, especially for stimulating vascular formation and reparation and tissue reparation and for immunotherapy. The invention also relates to nucleic acids which code for the inventive polypeptides, vectors containing these nucleic acids and antibodies targeting the polypeptides.

Description

Parapox virus-encoded vascular endothelial cell growth factor (PPV-VEGF)

description

The invention relates to new polypeptides obtainable from parapox viruses (PPV) with homologies to human VEGF proteins, which are able to stimulate endothelial cell proliferation. These PPV-VEGF polypeptides are suitable for pharmaceutical applications, in particular for stimulating vascularization, vascular repair, tissue repair and for immunotherapy. Furthermore, nucleic acids coding for the polypeptides according to the invention, vectors containing these nucleic acids and antibodies directed against the polypeptides are disclosed.

Angiogenesis involves the process of new formation of blood vessels from existing blood vessels. Angiogenetic processes are essential for normal development and differentiation of the vascular system. In the postembryonic stage, angiogenesis is only found in a few physiological processes such as the cyclic growth of the corpus luteum and endometrium, wound healing, the repair processes in tissue and organ regeneration, and in the pathological process of tumor growth (Beck and D'Amore, "Vascular Development: Cellular and Molecular Regulation ", FASEB J. 1 1: 365-373, 1997; Risau," Mechanisms of angiogenesis ", Nature 386: 671-674, 1 997).

Blood capillaries consist of endothelial cells and pericytes. These two cell types have the genetic information for the formation of new blood capillaries by capillary sprouting. This process is initiated by specific angiogenesis factors. Due to the great physiological importance of angiogenesis, various angiogenesis factors have been isolated, purified and characterized, among others aFGF, bFGF, EGF, TGF-σ, TGF-ß, PGE2, monobutyrin, HGF, TNF-σ, PD-ECGF, angiogenin, interleukin-8 and VEGF (Klagsbrun et al., "Regulators of Angiogenesis", Ann. Rev Physiol., 53: 217-239, 1,991).

VEGF (Vascular Endothelial Growth Factor) denotes a family of micro-heterogeneous growth factors with great specificity and selectivity with regard to the effect as mitogen for vascular endothelial cells (ED ₅₀ 2- 10 pM) (Ferrara et al., "The Vascular Endothelial Growth Factor Family of Polypeptides", J. Cellular Biochem., 47:21 1-21 8, 1,991). VEGF stimulates angiogenesis in various in vivo models, such as the "chick chorioallentoic membrane" (CAM) assay (Leung et al., "Vascular endothelial growth factor is a secreted angiogenic mitogenic. Science 246: 1306-1309, 1989) and the" rabbit cornea "assay (Philipps et al.," Vascular endothelial growth factor is expressed in rat corpus luteum ", Endocrinology, 1 27: 965-968, 1995), and in vitro models by forming capillary structures in three-dimensional collagen gels (Pepper et al., "Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro", Biochem. Biophys. Res. Commun. 1 89: 824-831, 1 992). VEGF stimulates in vascular endothelial cells also the expression of tissue-type plasminogen activator (tPA), PA inhibitor-1 (PAI-1) (Pepper et al., "Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor type 1 in microvascular endo thelial cell ", Biochim. Biophys. Res. Commun. 1 81: 902-908, 1991) and metal proteinases (Unemori et al., "Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells", J. Cell. Physiol. 1 53: 557-562, 1 992). Another function of VEGF is to increase vascular permeability, which is why the protein is also referred to as "Vascular Permeability Factor" (VPF) (Ferrara et al., "The Vascular Endothelial Growth Factor Family of Polypeptides", J. Cellular Biochem. , 47:21 1 -21 8 (1 991)). In addition, VEGF is said to have an immunomodulatory effect, since VEGF is the functional maturation of dendritic cells from CD34 + Stem cells inhibited (Gabrilovich et al., "Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells", Nature Med. 2: 1096-1 103, 1 996).

Of the various micro-heterogeneous human VEGF forms, VEGF ₁₆₅ is predominantly present in normal human tissues. VEGF ₁₆₅ is a glycosylated, cationic dimer of 46-48 kDa, which is formed from two identical 24 kDa subunits. VEGF ₁₆₅ is inactivated by sulfhydryl reducing agents, but it is resistant to acidic pH and heat. VEGF ₁₆₅ binds to the transmembrane receptors KDR and Flt-1 on endothelial cells, both of which have protein tyrosine kinase activity in the intracellular domain. While Flt-1 is bound with higher affinity than KDR, the mitogenic signal for endothelial cells appears to be mediated by KDR (Klagsbrun and D'Amore, "Vascular endothelial growth factor and its receptors", Cytokine Growth Factor Rev. 7: 259-270 , 1 996).

By differential splicing of transcripts of the human vegf gene, in addition to VEGF ₁₆₅ , the VEGF polypeptide variants VEGF ₁₂₁ , VEGF ₁₈₉ and VEGF ₂₀₈ can also be formed. While all human VEGF polypeptides have the property of stimulating endothelial cell proliferation, the various micro-heterogeneous forms differ in terms of size, the degree of low-affinity binding to heparin or the high-affinity binding to Flt-1 (Keyt et al., "The Carboxy Terminal Domain (1 1 1 - 165) of Vascular endothelial Growth Factor is Critical for its Mitogenic Potency "J. Biol. Chem. 271: 7788-7795, 1 996). In the meantime, other homologous genes and their gene products have been described in humans, including VEGF-B, VEGF-C and VEGF-D, which have different biological activities and expression patterns than the previously described VEGF polypeptides. Homologs of the designated human factors have also been described in various animal species. The parapox viruses include, among others, Orf viruses, which cause lip lip in sheep and goats and the ecthyma contagiosum in humans, and milking node viruses, which cause milking nodes in humans (for review articles see Mayr, A. and Büttner, M. "Milers's node, Bovine Papular Stomatitis, Ecthyma (Orf) Virus "In: Virus Infections of the Vertebrates, Vol. 3. Dinter, Z., Morein, B. (eds.) Pages 23-42, Elsevier, Amsterdam, 1990). These viruses infect keratinocytes in the skin and cause vasoproliferative processes, which in turn lead to mucosal proliferation. By DNA sequencing of comparable sections of the genome of the Orf virus isolates NZ2 and NZ7, two variants of a hypothetical gene were identified (DNA sequences of the open reading frames referred to here as NZ2vegf, see Fig. 1 a, or NZ7vegf, see Fig 1 b) with an open reading frame which has a certain homology to mammalian VEGF (Lyttle et al., "Homologs of Vascular Endothelial Growth Factor are encoded by the Poxvirus Orf Virus", J. Virol., 68:84 -92, 1994). However, the gene products of these genes and their biological activity are not known.

The availability of factors that can stimulate the growth of blood vessels is of great importance for the treatment of pathological conditions resulting from insufficient blood supply to tissues and organs (ischemia). Recombinant hVEGF ₁₆₅ (US Pat. No. 5,332,671) has already been successfully used in the rabbit animal model for the therapy of experimentally induced ischemia of an extremity (Takeshita et al, "Therapeutic angiogenesis: A Single intra-arterial bolus of vascular endothelial growth factor augments collateral vessel formation in a rabbit ischemic hindlimb model ", J. Clin. Invest 93: 662-670, 1994) and in a pig-animal model for the therapy of chronic myocardial ischemia (Harada et al.," Vascular endothelial growth factor improves coronary flow and myocardial function in chronically ischemic porcine hearts. Am. J. Physiol. 270: 1791-1802). Further VEGF variants were patented for similar therapeutic applications (US Patents No. 5,194,596, No. 5,607,918, No. 5,726,152). The different physical properties (e.g. binding to heparin, stability to proteolytic degradation) and biological activities (effect on vascular endothelial cells as a mitogen or as a permeability factor) of the different VEGF variants suggest that these factors have different therapeutic benefits for the treatment of these and others Clinical pictures are going to be. Since optimized therapeutic agents for use in humans have not yet been developed, there is therefore still a need to provide growth factors with the highest possible biological effectiveness and / or selective effect on the endothelium.

According to the present invention, a gene fragment was isolated from the genome of the Orf virus isolate D1701 (DNA sequence of the open reading frame, here referred to as D1701 vegf, see FIG. 1 (c)), which is homologous to the previously isolated hypohetic genes NZ2vegf (Fig. Ka)) and NZ7vegf (Fig. 1 (b)) from other Orf virus isolates (Lyttle et al., "Homologs of Vascular Endothelial Growth Factor are encoded by the Poxvirus Orf Virus", J. Virol. , 68: 84-92, 1994). Fig. 2 shows an alignment of the VEGF-homologous protein sequences of these genes with the protein sequences of mammalian forms of VEGF.

The expression of recombinant rPPV-VEGF in E.coli and its purification, as well as the native expression of PPV-VEGF in eukaryotic cells, enabled a potent growth factor for vascular endothelial cells to be demonstrated, which was surprisingly related to binding to the high-affinity surface receptors KDR and Flt-1 of human VEGF ₁₆₅ clearly differentiates: While both receptors bind with approximately the same affinity to the receptor KDR that transmits the mitogenic signal, rPPV-VEGF has no significant binding activity for the receptor Flt-1. A first aspect of the invention relates to an isolated polypeptide with the property of stimulating endothelial proliferation, containing an amino acid sequence selected from

(a) an amino acid sequence encoded by one of the nucleic acid sequences shown in Fig. 1 (a), (b) and (c),

(b) an amino acid sequence which contains at least 25 consecutive amino acid residues of one of the amino acid sequences from (a), or

(c) an amino acid sequence with an identity of at least 50% to one of the amino acid sequences from (a).

Another aspect of the invention relates to an isolated polypeptide with the property of stimulating endothelial proliferation, obtainable from parapox viruses, or a variant derived therefrom.

The invention provides a novel "vascular endothelial growth factor" which is encoded by a gene in the genome of parapox viruses (PPV-VEGF). This PPV is suitable for diagnostic and especially therapeutic applications, e.g. for the stimulation of vascular formation and repair, furthermore tissue repair, the production of artificial vessels and for immunotherapy. Furthermore, the provision of DNA vectors for the expression of PPV-VEGF is provided, in particular for the gene therapy of defects in vascularization, vascular repair and tissue repair.

The recombinant PPV-VEGF (rPPV-VEGF) presented in this invention is distinguished by a high activity with regard to the stimulation of the proliferation of human endothelial cells. In addition, the rPPV-VEGF according to the invention is distinguished from human VEGF ₁₆₅ by selective receptor binding. While human VEGF ₁₆₅ binds the receptors KDR and Flt-1 with high affinity, rPPV-VEGF only binds to the mitogenic signal-mediating receptor KDR. Another advantage of the invention lies in the method presented for the expression of rPPV-VEGF in E. coli, with which a sufficient amount of rPPV-VEGF can be produced for therapeutic use. The detection of a functional expression of PPV-VEGF by eukaryotic cells after transfection of an expression plasmid or after infection with whole PPV viruses also shows that the DNA sequence coding for PPV-VEGF can be used for gene therapy applications.

The nucleic acid sequences shown in FIGS. 1 (a), (b) and (c) code for polypeptides according to the invention. The corresponding nucleotide and amino acid sequences are also in SEQ ID NO. 1 and 2 (a), 3 and 4 (b) and 5 and 6 (c) respectively. The invention also encompasses polypeptides which have a partial amino acid sequence with at least 25, preferably at least 50 and particularly preferably at least 75 consecutive amino acid residues of one of these amino acid sequences or an amino acid sequence with an identity of at least 50%, preferably at least 60%, particularly preferably at least 75% and most preferably have at least 90% of any of these amino acid sequences.

A polypeptide containing an amino acid sequence is particularly preferably selected from

(a) an amino acid sequence different from that shown in Fig. 1 (c)

Nucleic acid sequence is encoded, (b) an amino acid sequence that is at least 25 consecutive

Contains amino acid residues of one of the amino acid sequences from (a), or (c) an amino acid sequence with an identity of at least 50% to one of the amino acid sequences from (a).

The polypeptide according to the invention is preferably obtained by recombinant expression of an exogenous nucleic acid sequence in a suitable one Host cell, for example a prokaryotic or eukaryotic host cell. When expressed in prokaryotic host cells such as E. coli, the polypeptide is obtained in unglycosylated form.

The nucleic acid sequence coding for the polypeptide is preferably selected from:

(a) the nucleic acid sequences shown in Fig. 1 (a), (b) and (c),

(b) nucleic acid sequences which correspond to a nucleic acid according to (a) in the context of the degeneration of the genetic code, and (c) nucleic acids which hybridize with a nucleic acid according to (a) or / and (b) under stringent conditions.

The term "stringent hybridization" according to the present invention is used as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1 .101-1 .104, 1 989). One preferably speaks of a stringent hybridization if, after washing for 1 hour with 1 X SSC and 0.1% SDS at 55 ° C., preferably at 62 ° C. and particularly preferably at 68 ° C., in particular for 1 h at 0.2 X SSC and 0.1% SDS at 55 ° C., preferably at 62 ° C. and particularly preferably at 68 ° C., a positive hybridization signal is also observed. It is further preferred if the nucleic acid sequence has an identity of at least 70%, particularly preferably of at least 80% and most preferably of at least 90% to one of the nucleic acid sequences shown in FIGS. 1 (a), (b) and (c).

Polypeptides according to the invention with the property of stimulating endothelial cell proliferation are obtainable from parapox viruses, in particular Orf viruses. In addition, the present invention also encompasses variants of such polypeptides which differ from the polypeptides obtainable from parapox viruses by substitutions, deletions and / or additions of individual amino acids or / and amino acid segments. These variants preferably contain at least 25, particularly preferably at least 50 and most preferably at least 75 consecutive amino acid residues of the natural viral polypeptides. Furthermore, it is preferred if the amino acid sequence of the variants has an identity of at least 50%, preferably at least 60%, particularly preferably at least 75% and most preferably at least 90% with the amino acid sequence of the natural viral polypeptides.

Yet another aspect of the present invention is an isolated nucleic acid, which is for a polypeptide as previously indicated, or a

Section of such a nucleic acid of preferably at least 20, particularly preferably at least 50 and most preferably at least

1 00 nucleotides encoded. The nucleic acid can be a DNA, RNA or also a nucleic acid analog. Nucleic acids according to the invention comprise nucleic acids obtainable from parapoxviruses, from them by recombinant ones

DNA techniques available nucleic acids and nucleic acids produced by chemical synthesis. Preferably the nucleic acid is a recombinant nucleic acid, i.e. it is linked to others

Nucleic acid sequences with which it is not naturally associated. The nucleic acid according to the invention is preferably free of further polypeptide-coding sequences from parapox viruses.

Yet another aspect of the present invention is a recombinant vector that contains at least one copy of a nucleic acid as previously indicated. This vector can be any prokaryotic or eukaryotic vector on which the nucleic acid according to the invention is preferably under the control of an expression signal (promoter, operator, enhancer, etc.). Preferred examples of vectors according to the invention are plasmids or viral vectors. Suitable prokaryotic vectors are described, for example, in Sambrook et al., Supra, chapters 1-4. Examples of eukaryotic vectors can be found in Sambrook et al., Supra, chapter 1 6. Yet another aspect of the present invention is a recombinant host cell that is transfected with a nucleic acid according to the invention or with a vector according to the invention. The term "transformation" in the sense of the present invention is to be understood in its broad meaning and also includes processes such as transfection or infection.

The host cell according to the invention can be a prokaryotic cell, in particular a gram-negative prokaryotic cell, e.g. be an E.coli cell. On the other hand, however, the cell can also be a eukaryotic cell, such as a fungal cell (e.g. yeast), an animal, human or a plant cell. Methods for transforming prokaryotic or eukaryotic cells with exogenous nucleic acids are familiar to the person skilled in the field of molecular biology.

In addition, the invention also relates to antibodies which are directed against a polypeptide according to the invention. These antibodies preferably have no cross-reactivity with human VEGF polypeptides. The antibodies of the invention can be obtained by immunizing experimental animals, e.g. Mice, rats or rabbits, with polypeptides according to the invention or sections thereof. Peptide sections which have no significant homology to human VEGF polypeptides are preferably used for the immunization. Polyclonal antisera or hybridoma cells capable of producing monoclonal antibodies can then be obtained in a known manner from the immunized experimental animals. In addition to polyclonal and monoclonal antibodies, fragments and derivatives of such antibodies are also detected by the present invention.

The polypeptide according to the invention is preferably produced by a process in which a host cell which is associated with a

Nucleic acid or transformed with a vector according to the invention provides, the host cell cultivated under conditions in which one Expression of the polypeptide takes place, and the polypeptide is recovered from the host cell and / or the culture medium. Methods for obtaining polypeptides according to the invention from prokaryotic and eukaryotic cells are described in detail in the examples of the present application.

An important aspect of the present invention is a pharmaceutical composition which contains, as active ingredient, a polypeptide according to the invention or a section thereof, preferably an at least 25 amino acids, particularly preferably at least 50 and most preferably at least 75 amino acids, a nucleic acid according to the invention or a section thereof or a vector according to the invention or an antibody according to the invention optionally together with pharmaceutically customary carriers, auxiliaries and additives. The composition according to the invention can be used for diagnostic applications and in particular for therapeutic applications.

Diagnostic applications include methods for the detection of the polypeptide according to the invention, nucleic acids coding therefor or antibodies directed against the polypeptide in biological samples. Such detection methods are usually carried out in the form of immunoassays or nucleic acid hybridization assays. Such processes are familiar to the person skilled in the art.

Therapeutic applications for the pharmaceutical composition according to the invention include the stimulation of angiogenesis, the stimulation of tissue repair or the modulation of the immune response, for example the stimulation of endothelial cell proliferation or the inhibition of the functional differentiation of dendritic cells. The composition according to the invention can be administered according to protocols as are known, for example, for human VEGF polypeptides, for example by parenteral administration of the polypeptide. The is also of importance Use of the pharmaceutical composition for DNA vaccination or for gene therapy. Known vector systems can be used for this purpose, the use of which is known for such purposes. In addition, infectious parapox viruses, in particular non-pathogenic variants thereof, can also be used as vector systems.

In addition to the polypeptides, nucleic acids or antibodies according to the invention, the pharmaceutical composition according to the invention can also contain one or more active ingredients. Examples of such active ingredients are known angiogenesis factors as mentioned previously.

The present invention is further illustrated by the following examples, figures and sequence listings. Show it:

Figure 1 DNA sequences of the reading frame of a gene with homology to animal VEGF growth factors, which are encoded in the genome of the Orf virus isolates NZ2 (NZ2vegf; a), NZ7 (NZ7vegf; b) and D1 701 (D1701 vegf; c).

Figure 2 shows an alignment of the protein sequences of VEGF homologues from Orf virus D1 701 (D1 701), NZ2 (NZ2, Genbank Accession No. S67520), NZ7 (NZ7, Genbank Accession No. S67522) and of human VEGF1 21 (hVEGFI 21 , Genbank Accession No. M32977), sheep VEGF (oVEGF, Genbank Accession No. X89506), human VEGF165 (hVEGF1 65, Genbank Accession No. M32977), pigs VEGF (pVEGF, Genbank Accession No. X81 380), rats VEGF (rVEGF , Genbank Accession No. M 32167) and human VEGF-B (hVEGF-B, Genbank Accession No. U48801). The augmentation was carried out with the program Multialign (Corpet, "Multiple sequence alignment with hierarchical clustering", Nucl. Acids Res. 16: 10881-10890, 1998). Figure 3 shows the plasmid map of pCD371, a plasmid of approximately 6550 bp in size for the recombinant expression of PPV-VEGF in E. coli.

Figure 4 shows the amino acid sequence of rPPV-VEGF before proteolytic processing with thrombin. The amino acids of the vector-encoded N-terminal peptide are underlined, the proteolytic cleavage site for thrombin is marked by an asterisk, the amino acid sequence from the open reading frame of PPV-VEGF is shown in bold.

Figure 5 shows the concentration-dependent stimulation of endothelial cell proliferation (HUVECs) by rPPV-VEGF.

Figure 6 shows the heat resistance (heated, 25 min 100 ° C) and proteinase K sensitivity (Prot. K, digestion of 50 ng rPPV-VEGF with 25 ng / ml proteinase K for 3 h at 37 ° C, then inactivation of the proteinase K for 25 min at 100 ° C) the activity of rPPV-VEGF to stimulate endothelial cell proliferation (HUVECs)

Figure 7 shows the inhibition of stimulation of endothelial cell proliferation (HUVECs) by rPPV-VEGF in the presence of soluble Flt-1 receptor (sFLT-1; 2.7 μg / ml).

Figure 8 shows the plasmid map of pCD379, a plasmid for the expression of PPV-VEGF in eukaryotic cells.

9 shows the stimulation of endothelial cell proliferation (HUVECs) by conditioned cell culture supernatants from COS-7 cells, which were treated with the PPV-VEGF expression plasmid pCD379 or the empty one

Expression vector pCB6-Bam were transfected. 10 shows the stimulation of endothelial cell proliferation (HUVECs) by cell culture supernatants from BKKL3A cells which had been infected with an MOI = 1 of the OV virus D1701 for 24 h.

Figure 1 1 the in vitro angiogenesis by rPPV-VEGF.

Figure 1 2 the in vivo angiogenesis by rPPV-VEGF in comparison to human VEGF ₁₆₅ in the "Rabbit Cornea Assay".

Figure 1 3 shows a displacement assay with ¹²⁵ l-labeled human VEGF ₁₆₅ for determining the specificity of rPPV-VEGF and _VEGF-165 1 Flt (left) and KDR (right) for endothelial cell receptors.

SEQ ID NO. 1/2: The nucleotide sequence of the vegf gene from the Orf virus isolate NZ2 and the corresponding amino acid sequence.

SEQ ID NO. 3/4: The nucleotide sequence of the vegf gene from the Orf virus

Isolate NZ7 and the corresponding amino acid sequence.

SEQ ID NO. 5/6: The nucleotide sequence of the vegf gene from the Orf virus isolate D1701 and the corresponding amino acid sequence. Examples

1 . Cloning of the D1701vegf gene from the genome of the Orf virus D1701

The gene D1 701 vegf was amplified from genomic DNA of the Orf virus D1701 using the polymerase chain reaction (standard technique according to Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Edition) Cold Spring Harbor Laboratory Press, 1 989). For this purpose, the oligonucleotide primers 5'-GGG TGA GAA TTC ATG AAG TTT CTC GTC GGC-3 'and 5'- AAA GTT GGA TCC CTA GCG GCG TCT TCT GGG-3' which immediately before the ATG start codon an EcoRI- Interface and introduce a BamHI interface immediately after the TAG stop codon. The approximately 420 bp amplificate was cloned into the vector pSPT18 (Boehringer Mannheim) via the introduced EcoRI and BamHI cleavages by standard techniques, whereby the plasmid pVEGF was formed. The 1701 vegf gene cloned in pVEGF was further cloned into the vector pSK ^" (Stragene, Heidelberg) via the EcoRI and BamHI cleavage sites, whereby the plasmid pCD325 was formed. The cloned DNA was sequenced. The DNA sequence of the encoded open reading frame of the D1 701 egf gene is shown in Figure 1 c.

2. Cloning of the D1701vegf gene in vectors for the recombinant expression of a fusion protein in E. coli

The recombinant expression of rPPV-VEGF was carried out in derivatives of the vector pET-15b (Novagen, Madison, Wisconsin). This IPTG-inducible expression vector based on T7 polymerase enables the expression of fusion proteins which carry a proteolytically cleavable peptide with a hexa-histidine sequence for the affinity purification of the recombinant protein on nickel chelate columns at the N-terminal. The fused portion of the D1701 vegf gene was truncated 20-amino acids at the N-terminal because of this roughly corresponds to the processing of the native protein when the signal peptide is split off during the co-translational translocation in eukaryotic cells (Fig. 4). For the cloning of the expression plasmid pCD369, the vector pET-15b was opened with Ndel, the overhanging ends were filled in with Klenow polymerase and then the BamHI site located at one end was cut with BamHI. An approximately 345 bp gene fragment of D1 701 vegf was isolated by Mvnl / BamHI digestion of the plasmid pCD325 and directionally cloned into the vector pET-1 5b prepared in the previous step, whereby the plasmid pCD369 was reconstituted (Fig. 3a).

However, the introduction of pCD369 into the E. coli expression strain BL21 (DE-3) (Novagen, Madison, Wisconsin) alone did not yet allow expression of the recombinant fusion protein. For this purpose, the plasmid pSB1 61 (Schenk et al., "Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ^Ar9 tRNAs) had to be introduced, which causes the overexpression of a rare ^Ar9 tRNA (argU). As a result, genes can be overexpressed in E. coli which, like many eukaryotic genes, contain a critical number of arginine codons (AGG / AGA), which are rare in E. coli (Brinkmann et al., "High-level expression of recombinant genes in Escherichia coli is dependent on the availabilitiy of the dnaY gene product ", Gene 85: 109-1 14, 1 989). In order to improve the expression of the recombinant pPPV-VEGF protein and to improve the stability of the expression plasmid, plasmids pCD369 and pSB1 61 constructed the chimeric plasmid pCD371, which combines the positive features of the two plasmids and largely excludes their disadvantages: (1) a Lac ^q repressed, IPTG-inducible T7 polymerase Common expression system for the overexpression of the fusion protein according to Fig. 4, (2) expression of the rare ^Ar9 tRNA (argU) as a prerequisite for overexpression of the latter fusion protein and (3) a kanamycin resistance as a more suitable resistance marker compared to the ampicillin resistance in pCD369. For the construction of the expression plasmid pCD371 (Fig. 3b) pSB1 61 was linearized with Nhel and the overhanging ends were filled in with Klenow polymerase. pCD369 was digested with Nrul / Sspl and a 2844 bp fragment with the lac ^q gene and the D1701 vegf gene was cloned into the vector pSB1 61 linearized in the previous step. pCD371 was then transformed into the expression strain BL21 (DE-3). This expression strain was found to be stable and suitable for the expression of large amounts of the rPPV-VEGF fusion protein in E. coli.

3. Recombinant expression of the rPPV-VEGF fusion protein in E. coli and its purification

For the expression of the rPPV-VEGF fusion protein in E. coli, the strain BL21 (DE-3, pCD371) was grown from a glycerol stock in LB medium with 30 μg / ml kanamycin for 1 6 h in shaking culture at 37 ° C. Then 2 liters of the same medium were inoculated 1: 100 with the culture and incubated to an optical density (OD550nm) of 0.3 under the same conditions. After adding IPTG (1 mM final concentration), the mixture was incubated for a further 2 h. The bacteria were then pelleted by centrifugation and resuspended in digestion buffer (5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 7.9) and disrupted at 4 ° C. by ultrasound. After centrifugation for 30 min at 1,700 rpm, 4 ° C., soluble rPPV-VEGF from the supernatant was determined using the hexa-histidine tag by affinity chromatography on a Ni-chelate column according to the manufacturer's instructions (Novagen, Madison, Wisconsin, protocol for native cleaning) and separated from contaminating substances by washing with at least 10 column volumes.

After elution, the protein solution was concentrated by ultrafiltration using Centricon columns according to the manufacturer's instructions (Amicon, Beverly, MA). Another cleaning step of rPPV-VEGF was carried out by

Gel filtration over Superose 1 2 h in PBS buffer, resulting in dimers for the most part could be separated from remaining monomers. Then the histidine day was split off by digestion with 1 U biotinylated thrombin / mg fusion protein for 6.5 h at room temperature in accordance with the manufacturer's instructions (Novagen, Madison, Wisconsin) and the biotinylated thrombin was adsorbed on streptavidin-Sepharose. The cleaved histidine tag was adsorbed on a Ni chelate column according to the manufacturer's instructions (Novagen, Madison, Wisconsin). After the column flow was again concentrated by ultrafiltration, a last gel filtration step was carried out over a Sephacryl S-200 column in PBS buffer. The homogeneity of the eluted rPPV-VEGF was demonstrated by SDS-PAGE and subsequent silver staining using standard techniques. Protein determination with the Bradford reagent (Biorad) was carried out according to the manufacturer's instructions. Endotoxin determinations were carried out using the Limulus Amebocyte Lysate (LAD Coattest (Chromogenix, Mölndal, Sweden) according to the manufacturer's instructions.

4. Test of endothelial cell proliferation by rPPV-VEGF and PPV-VEGF

The stimulation of the proliferation of primary endothelial cells from the vein of the human umbilical cord ("Human umbilical vein endothelial cells" hereinafter referred to as HUVECs) is the usual method for demonstrating the mitogenic effect of VEGF growth factors on endothelial cells. HUVECs were developed according to Dehio et al. , ("Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internation of the bacterial aggregate by a unique structure, the invasome", J. Cell Science 1 10: 2141-21 54, 1 997) prepared and cultivated and used from passage 4 to 9 for proliferation assays. For this, HUVECs with a density of 50,000 cells / ml in gelatin-coated coating with (0.2% solution at 37 ° C for 30 min) 24 well plates were sown in medium 1 99 with 10% decomplemented FCS and for 1 6 h at 37 ° C / 5% CO ₂ incubated. Then the medium was exchanged for the same medium with the test substance and after a further 72 h incubation of the cells at 37 ° C./5% CO ₂ , the cell number was determined by counting a defined microscopic field. Medium without further additives was used as the reference sample to determine the proliferation factor; the proliferation factor of a sample results from the quotient of the cell numbers based on this reference sample.

With this test, the activity of rPPV-VEGF and human VEGF _{165 was} compared (Fig. 5) and the sensitivity of rPPV-VEGF to heating and proteinase K digestion was tested (Fig. 6). In order to demonstrate the specificity of the mitogenic signal of rPPV-VEGF for endothelial cells, a comparable proliferation test was also carried out for human microvascular endothelial cells (HDMVEC), human fibroblasts (NHDF) and human smooth muscle cells (SMC) (Fig. 7).

5. Cloning of the D1701vegf gene into a eukaryotic expression vector

For the expression of PPV-VEGF in eukaryotic cells, the gene D1701 vegf was cloned into the expression vector pCB6-Bam (Fig. 8a). pCB6-Bam is a plasmid 61 89 bp in length, which has the following functional sequence elements: the origin of replication ColE1 and the ampicillin resistance-coding gene bla, which enables cloning and plasmid preparation in E. coli, an expression cassette for the neo gene, which confers resistance to geniticin in transfected eukaryotic cells; a cytomegalovirus (CMV) promoter, followed by a polylinker and an hGII polyadenylation site for expression of any gene in eukaryotic cells by inserting the coding sequences into the polylinker. The reading frame of the D1701 vegf gene cloned in pVEGF or pCD325 is framed directly by the previously recombinantly introduced EcoRI or BamHI cleavage sites. For the efficient Expression of D1 701 vegf in eukaryotic cells, however, appears to be of importance to a few nucleotides of the viral sequence upstream from the translation start point. For this reason, 22 nucleotides of the original viral sequence in front of the ATG start codon were reintroduced using suitable primers. For the polymerase chain reaction using standard techniques, the plasmid pCD325 was used as a template and the oligonucleotides 5'-GGG GAA GCT TAG TTT TAA GGG TGA GGC GCC ATG AAG TTT CTC GTC GGC ATA CTG-3 'and 5'-CGC GGA TCC CTA GCG GCG TCT TCT G-3 'used as an amplification primer. The amplificate was cloned into the vector pCB6-Bam via standard HindII and BamHI interfaces, which resulted in the formation of the plasmid pCD379 (FIG. 8b).

6. Expression of PPV-VEGF in eukaryotic cells

Plasmid DNA of the eukaryotic expression vector pCD371 for transfection experiments was prepared with the Qiagen Midi Kit (Qiagen, Hilden) according to the manufacturer's instructions. COS-7 cells were obtained using the Ca-phosphate coprecipitation technique (standard technique according to Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Edition) Cold Spring Harbor Laboratory Press, 1 989) with the PPV-VEGF expression plasmid pCD379 or the empty expression vector pCB6-Bam. From 24 h to 72 h after the transfection, medium (M 1 99/10% decomplemented FCS) was conditioned on the transfected cells. After harvesting the culture supernatants, they were centrifuged for 1 5 min and then filtered through 0.22 μm filters. Then dilutions thereof were tested in a HUVEC proliferation assay according to Example 4 (Fig. 9). 7. Expression of PPV-VEGF by PPV-infected cells

BKKL3A cells, or other cell cultures from the calf kidney or lung, or from the skin of sheep, cattle or humans were infected by standard techniques with Orf viruses and a multiplicity of the infection from 0.1 to 10 for 4-48 h . Cell culture supernatants are then filtered through 0.1 μm filters, which themselves retain the virus particles, and tested in a HUVEC proliferation assay in accordance with Example 4 (FIG. 10).

8. In vitro angiogenesis by rPPV-VEGF

As an in vitro angiogenesis assay, a so-called "sprouting assay" with endothelial cell spheroids in the collagen gel was carried out, a modification of the "microcarrier-bead" angiogenesis assay (Nehls and Drenckhahn, 1,995). For this purpose, endothelial cell spheroids were produced as described by Korff and Augustin (1 998, J. Cell. Biol. 143, 1 341-1 352). Figure 1 1 shows the activity of rPPV-VEGF in this assay compared to human VEGF ₁₆₅ .

9. In vivo angiogenesis by rPPV-VEGF

As an in vivo angiogenesis assay, the "rabbit cornea" assay in the cornea of albino rabbits was carried out according to Ziehe et al. (1 994, J. Clin. Invest., 94, 2036-2044 and 1 997, Lab. Invest. 76, 51 7-531) carried out in parallel with human VEGF ₁₆₅ (Fig. 1 2).

10. Binding of rPPV-VEGF to VEGF Receptors (KDR or Flt-1)

For the binding studies, ¹²⁵ l-labeled human VEGF ₁₆₅ with different concentrations of cold human VEGF ₁₆₅ or rPPV-

VEGF in RPMI mixed with 20 mM Hepes and 0.1% gelatin. To 3T3-

Fibroblasts that had been stably transfected with Flt-1 or PAE cells, expressing the KDR, this solution was given and incubated for 3 hours on ice. After 4 washing steps with RPMI medium with 0.1% gelatin to remove unbound ¹²⁵ I-VEGF ₁₆₅ , the cells were lysed with 0.1% SDS and the cpm of the released ¹²⁵ I-VEGF ₁₆₅ in the gamma counter (Packard ) determined (Fig. 13).

Claims

Expectations

1 . Isolated polypeptide with the property of stimulating endothelial proliferation, containing an amino acid sequence selected from

2. Polypeptide according to claim 1, comprising an amino acid sequence selected from

(a) an amino acid sequence encoded by the nucleic acid sequence shown in Fig. 1 (c),

3. Polypeptide according to one of claims 1 or 2, characterized in that it is unglycosylated.

4. Polypeptide according to one of claims 1 to 3, characterized in that it is produced by recombinant expression of an exogenous nucleic acid sequence in a suitable host cell.

5. Polypeptide according to one of claims 1 to 4, characterized in that the nucleic acid sequence is selected from:

(a) the nucleic acid sequences shown in Fig. 1 (a), (b) and (c),

(b) nucleic acid sequences which correspond to a nucleic acid according to (a) in the context of the degeneration of the genetic code, and

(c) Nucleic acids which hybridize under stringent conditions with a nucleic acid according to (a) or / and (b).

6. Isolated polypeptide with the property of stimulating endothelial cell proliferation, obtainable from parapox viruses, or a variant derived therefrom.

7. Polypeptide according to claim 6, characterized in that it is obtainable from Orf viruses.

8. An isolated nucleic acid encoding a polypeptide according to any one of claims 1 to 7 or a portion thereof.

9. Recombinant vector containing at least one copy of a nucleic acid according to claim 8.

10. Vector according to claim 9, characterized in that the nucleic acid is in operative association with an expression signal.

1 1. Vector according to one of claims 9 or 10, characterized in that it is a plasmid.

1 2. Vector according to one of claims 9 or 10, characterized in that it is a viral vector.

1 3. Vector according to one of claims 9 to 12, characterized in that it is free of further polypeptide-coding sequences from parapox viruses.

14. Recombinant host cell, characterized in that it is transformed with a nucleic acid according to claim 8 or a vector according to one of claims 9 to 13.

15. Antibody against a polypeptide according to one of claims 1 to 7.

16. Antibody according to claim 15, characterized in that it has no cross-reactivity with human VEGF polypeptides.

17. A method for obtaining a polypeptide according to any one of claims 1 to 7, characterized in that a host cell containing a nucleic acid according to claim

8 or a vector is transformed according to any one of claims 9 to 13, provides the host cell cultivated under conditions, in which expression of the polypeptide takes place and the polypeptide is obtained from the host cell and / or the culture medium.

1 8. Pharmaceutical composition, the active ingredient being a polypeptide according to one of claims 1 to 7 or a section thereof, a nucleic acid according to claim 8 or a section thereof, a vector according to one of claims 9 to 1 3 or an antibody according to claim 1 5 or 1 6 optionally together with pharmaceutically customary carriers, auxiliaries and additives.

1 9. Pharmaceutical composition according to claim 1 8 for diagnostic applications.

20. Pharmaceutical composition according to claim 1 8 for therapeutic applications.

21. Pharmaceutical composition according to claim 20 for modulating the immune response, for stimulating angiogenesis or for stimulating tissue repair.

22. Pharmaceutical composition according to claim 20 or 21 for stimulating endothelial cell proliferation or for inhibiting the functional differentiation of dendritic cells.

23. Pharmaceutical composition according to one of claims 20 to 22 for DNA vaccination and gene therapy.

24. Pharmaceutical composition according to one of claims 1 9 to 23, characterized in that it contains one or more other active ingredients.

5. Pharmaceutical composition according to claim 24, characterized in that it contains one or more further angiogenesis factors or nucleic acids coding therefor.