KR20010034576A - Vascular endothelial growth factor 2 - Google Patents

Abstract

Described herein are human VEGF-2 polypeptides, biologically active, diagnostic or therapeutically useful fragments thereof, homologues or derivatives thereof, and DNA (RNA) encoding such VEGF-2 polypeptides. Also provided are methods of making said polypeptides by recombinant techniques, and antibodies and antagonists against said polypeptides. The polypeptides and polynucleotides can be used therapeutically to stimulate wound healing and repair vascular tissue. Also provided are methods of using the antibodies and antagonists to inhibit tumor angiogenesis to inhibit tumor growth, inflammation, diabetic retinopathy, rheumatoid arthritis and psoriasis.

Description

Vascular endothelial growth factor 2

The present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, and methods of making such polynucleotides and polypeptides. Polypeptides of the invention have been identified as members of the vascular endothelial growth factor family. More particularly, the polypeptide of the present invention is human vascular endothelial growth factor 2 (VEGF-2). The present invention also relates to inhibiting the action of the polypeptide.

Formation of new blood vessels or angiogenesis is essential for embryonic development, continuous growth and repair of manipulation. Angiogenesis is also an integral part of certain pathological diseases such as neoplasia (ie tumors and glioma). Abnormal angiogenesis is associated with other diseases such as inflammation, rheumatoid arthritis, psoriasis and diabetic retinopathy (Flokman, J. and Klagsbrun, M. Science 235: 442-447 (1987)).

Both acidic and basic fibroblast growth factor molecules are mitotics for endothelial cells and other cell types. Angiotropins and angiogenins can induce angiogenesis, but their function is unclear. Folkman, J., Cancer Medicine, Lea and Febiger Press, pp. 153-170 (1993). A highly selective mitotic material for vascular endothelial cells is the factor vascular endothelial growth factor or VEGF, also known as vascular permeability factor (VPF). Ferrara, N. et al., Endocr. Rev. 13: 19-32 (1992).

Vascular endothelial growth factor is a secreted angiogenic mitogenic substance whose target cell specificity is believed to be limited to vascular endothelial cells. The rat VEGF gene was identified and its expression patterns in embryogenicity were analyzed. Sustained expression of VEGF was observed in epithelial cells adjacent to the lancet-like endothelial, such as choroid plexus and renal glomeruli. This data is consistent with the role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation (Breier, G. et al., Development 114: 521-532 (1992)).

VEGF has sequence homology with human platelet-derived growth factors PDGFa and PDGFb. Leung, D.W. et al., Science 246: 1306-1309 (1989). The homology rates are 21% and 23%, respectively. Eight cysteine residues involved in disulfide bond formation are purely conserved in these proteins. Although they are similar, there are specific differences between VEGF and PDGF. PDGF is a major growth factor for connective tissue, but VEGF is highly specific for endothelial cells. Another sliced mRNA has been identified for all of VEGF, PLGF and PDGF, and these different slicing products differ in terms of biological activity and receptor-binding specificity. VEGF and PDGF act as homo-dimer or hetero-dimer and are associated with receptors that induce intrinsic tyrosine kinase activity involving receptor dimerization.

VEGF has four different forms of 121, 165, 189 and 206 amino acids due to another slicing. VEGF121 and VEGF165 are soluble and can enhance angiogenesis, while VEGF189 and VEGF206 bind to heparin containing proteoglycans on the cell surface. Temporal and spatial expression of VEGF has been correlated with physiological proliferation of blood vessels. Gajdusek, C.M., and Carbon, S.J., Cell Physiol. 139: 570-579 (1989); McNeil, P.L., et al., J. Cell. Biol. 109: 811-822 (1989). Its high affinity binding site is localized only on endothelial cells in tissue sections. Jakeman, L.B., et al., Clin. Invest. 89: 244-253 (1989). This factor can be isolated from pituitary cells and several tumor cell lines and is embedded in some human glioma (Plate, K.H., Nature 359: 845-848 (1992)). Interestingly, expression of VEGF121 or VEGF165 confers the ability to form tumors in nude mice to Chinese hamster ovary cells. Ferrara, N. et al., J. Clin. Invest. 91: 160-170 (1993). Inhibition of VEGF function by anti-VEGF monoclonal antibodies has been shown to inhibit tumor growth in immunodeficient mice (Kim, K.J., Nature 362: 841-844 (1993)). In addition, dominant-negative mutants of the VEGF receptor have been found to inhibit the growth of glioma in mice.

Vascular permeability factor (VPF) has also been found to be associated with sustained microvascular permeability to plasma proteins even after wound progression, a characteristic aspect of normal trauma healing. This suggests that VPF is an important factor in trauma healing. Brown, L.F. et al., J. Exp. Med. 176: 1375-1379 (1992).

Expression of VEGF is high in vascular tissues (eg, lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VEGF has also been shown to induce angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissues, especially vascular well tissues, VEGF has been proposed for use in enhancing vascular tissue repair (eg, in atherosclerosis).

US Pat. No. 5,073,492 (Dec. 19, 1991) to Chen et al. Synergistically enhances endothelial cell growth under such circumstances, including adding VEGF, effectors and serum-derived factors to the appropriate environment. The method is described. In addition, vascular endothelial cell growth factor C subunit DNA was prepared by polymerase chain reaction technology. Such DNA encodes certain proteins that may exist as hetero-dimer or homo-dimer. This protein is a mammalian vascular endothelial cell mitotic material and, as such, is useful for enhancing vascular development and repair, as described in EP 92302750.2 (September 30, 1992).

Summary of the Invention

Polypeptides of the invention have been presumably identified as novel vascular endothelial growth factors based on amino acid sequence homology with human VEGF.

According to one aspect of the present invention, novel mature polypeptides, and biologically active fragments thereof, and diagnostic, therapeutically useful fragments, homologs and derivatives are provided. Polypeptides of the invention are of human origin.

According to another aspect of the present invention, a bacterial host as shown in SEQ ID NO: 97149 on May 12, 1995 or ATCC Accession No. 75698 on March 4, 1994, or the amino acid sequence set forth in SEQ ID NO: 2 or 4, respectively An isolated nucleic acid molecule is provided that comprises a polynucleotide encoding a full length or truncated VEGF-2 polypeptide having an amino acid sequence encoded by a cDNA clone deposited within.

The present invention also relates to biologically active fragments of VEGF-2 and fragments, homologs and derivatives that are diagnostically or therapeutically useful.

According to another aspect of the invention, recombinant prokaryotic and / or eukaryotic host cells containing nucleic acid sequences encoding polypeptides of the invention are cultured under conditions that enhance expression of the protein and continuously recover such protein. There is provided a method of producing the polypeptide by recombinant technology comprising the step of.

According to a further aspect of the invention, the polypeptide, or such polypeptide, is encoded for therapeutic purposes, e. A method of using a polynucleotide is provided. In particular, for the treatment of peripheral arterial diseases such as critical lichen ischemia and coronary disease, methods are provided which utilize such polypeptides or polynucleotides encoding such polypeptides.

In addition, according to another aspect of the present invention, there are provided antibodies to said polypeptides and methods of making such polypeptides.

In addition, according to another aspect of the present invention, it inhibits the action of the polypeptide, for example, inhibits tumor growth by preventing tumor angiogenesis, and prevents diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis. An antagonist of the polypeptide is provided that can be used to treat it.

In addition, according to another aspect of the invention, a nucleic acid probe is provided comprising a nucleic acid molecule of sufficient length to specifically hybridize with a nucleic acid sequence of the invention.

In addition, according to another aspect of the invention, a method is provided for diagnosing a disease associated with or susceptible to a mutation in a nucleic acid sequence of the invention and a protein encoded by such nucleic acid sequence.

According to a further aspect of the invention there is provided a method for using said polypeptide, or polynucleotide encoding such polypeptide, for in vitro purposes relating to scientific research, DNA synthesis and construction of DNA vectors.

These and other aspects of the invention should be apparent to those skilled in the art from the teachings herein.

The following drawings illustrate aspects of the invention and do not limit the scope of the invention, which is encompassed by the claims.

1A-1E show the full length nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO: 2) sequences of VEGF-2. The polypeptide comprises approximately 419 amino acid residues, of which about 23 represent the leader sequence. Standard uppercase abbreviations for amino acids are used. Sequencing is performed using a Model 373 automated DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy is expected to exceed 97%.

2A-2D show the nucleotide (SEQ ID NO: 3) and deduced amino acid (SEQ ID NO: 4) sequences of the cleaved biologically active forms of VEGF-2. The polypeptide contains approximately 350 amino acid residues, of which the first 24 amino acids represent the leader sequence.

3A and 3B illustrate amino acid sequence homology between PDGFa (SEQ ID NO: 5), PDGFb (SEQ ID NO: 6), VEGF (SEQ ID NO: 7), and VEGF-2 (SEQ ID NO: 4). The boxed portion indicates the conserved sequence and the positions of the eight conserved cysteine residues.

4 is a table showing the homology between PDGFa, PDGFb, VEGF and VEGF-2.

5 shows the presence of VEGF-2 mRNA in human breast tumor cell lines.

6 shows the results of Northern blot analysis of VEGF-2 in human adult tissues.

Figure 7 shows the SDS-PAGE gel photograph after in vitro transcription, translation and electrophoresis of the polypeptide of the present invention. Lane 1: ¹⁴ C and rainbow MW marker; Lane 2: FGF control; Lane 3: VEGF-2 produced by the M13-reverse and forward primers; Lane 4: VEGF-2 produced by the M13-rear and VEGF-F4 primers; Lane 5: VEGF-2 produced by the M13-rear and VEGF-F5 primers.

8A and 8B show photographs of SDS-PAGE gels. VEGF-2 polypeptide was expressed in a baculovirus system consisting of Sf9 cells. Cell media and proteins from the cytoplasm are analyzed by SDS-PAGE under non-reducing (FIG. 8A) and reducing (FIG. 8B) conditions.

9 shows an SDS-PAGE gel photograph. A medium from Sf9 cells infected with the nucleic acid sequence of the present invention is precipitated. Resuspended precipitate is analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue.

10 shows an SDS-PAGE gel photograph. VEGF-2 is purified from the media supernatant and analyzed by SDS-PAGE in the presence or absence of the reducing agent b-mercaptoethanol and stained with Coomassie Brilliant Blue.

FIG. 11 shows reverse phase HPLC analysis of VEGF-2 purified using RP-300 column (0.21 × 3 cm, Applied Biosystems, Inc.). The column is equilibrated with 0.1% trifluoroacetic acid (solvent A) and the protein is eluted with a 7.5 minute gradient of 0-60% solvent B, consisting of acetonitrile containing 0.07% TFA. This protein eluate is monitored for absorbance at 215 nm ("red" line) and 280 nm ("blue" line). The percentage of solvent B is shown by the "green" line.

12 is a bar graph illustrating the effect of partially-purified VEGF-2 protein on the growth of vascular endothelial cells as compared to basic fibroblast growth factor.

13 is a bar graph illustrating the effect of purified VEGF-2 protein on the growth of vascular endothelial cells.

14 shows expression of VEGF-2 mRNA in human fetus and adult tissues.

15 depicts expression of VEGF-2 mRNA in human primary cultured cells.

16 depicts transient expression of VEGF-2 protein in COS-7 cells.

FIG. 17 depicts VEGF-2 stimulated proliferation of human umbilical vein endothelial cells (HUVEC).

18 shows VEGF-2 stimulated proliferation of skin microvascular endothelial cells.

19 depicts the stimulatory effect of VEGF-2 on proliferation of microvascular, umbilical cord, endometrium, and bovine aortic endothelial cells.

20 shows inhibition of PDGF-induced vascular (human aortic) smooth muscle cell proliferation.

21 depicts the stimulation of migration of HUVECs and bovine microvascular endothelial cells (BMEC) by VEGF-2.

FIG. 22 depicts nitric oxide release stimulation of HUVECs by VEGF-2 and VEGF-1.

FIG. 23 shows inhibition of macrophages of microvascular endothelial cells (CADMEC) by VEGF-2.

24 depicts stimulation of angiogenesis by VEGF, VEGF-2 and bFGF in the CAM assay.

FIG. 25 shows the restoration of certain parameters in ischemic paper by VEGF-2 protein (FIG. 25, top panel) and exposed expression plasmid (FIG. 25, middle panel): BP ratio (FIG. 25A); Blood flow and blood flow reserve (FIG. 25B); Angiographic score (FIG. 25C); Capillary Density (FIG. 25D).

26A-26G depict the ability of VEGF-2 to affect diastolic blood pressure in spontaneous hypertensive rats (SHR). 26A and B show dose-dependent decreases in diastolic blood pressure achieved with VEGF-2. 26C and d show the decrease in mean arterial pressure (MAP) observed with VEGF-2. Panel E depicts the dose-increasing effect of VEGF-2 on mean arterial pressure (MAP) in SHR rats. Panel F depicts the effect of VEGF-2 on diastolic blood pressure in SHR rats. Panel G depicts the effect of VEGF-2 on diastolic blood pressure in SHR rats.

27 shows inhibition of VEGF-2N- and VEGF-2-induced proliferation.

28 is a schematic of the pHE4a expression vector (SEQ ID NO: 16). The locations of the kanamycin resistance marker gene, multiple cloning site linker region, oriC sequence and lacIq coding sequence are indicated.

Figure 29 depicts the nucleotide sequence of the regulatory element of the pHE4a promoter (SEQ ID NO: 17). Two lac operator sequences, Shine-Delgarno sequence (S / D) and terminal HindIII and NdeI restriction sites (in italics) are indicated.

According to an aspect of the invention there is provided an isolated nucleic acid molecule comprising a polynucleotide encoding a VEGF-2 polypeptide (SEQ ID NO: 2) having an inferred amino acid sequence of FIG. 1, determined by sequencing a particular cloned cDNA. do. The nucleotide sequence set forth in SEQ ID NO: 1 is obtained by sequencing the cDNA clone deposited as ATCC No. 97149 in the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) on May 12, 1995.

According to another aspect of the invention, an isolated nucleic acid comprising a polynucleotide encoding a truncated VEGF-2 polypeptide (SEQ ID NO: 4) having the inferred amino acid sequence of FIG. 2, determined by sequencing a particular cloned cDNA. Molecules are provided. The nucleotide sequence set forth in SEQ ID NO: 3 is obtained by sequencing the cDNA clone deposited as ATCC No. 75698 in the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) on March 4, 1994.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein are determined using an automated DNA sequencing device (eg, model 373 from Applied Biosystems, Inc.) and determined by the DNA molecule determined herein. All amino acid sequences of the encoded polypeptide are expected by translation of the DNA sequence determined as above. Thus, as is known in the art for any DNA sequence determined by the automated approach, all nucleotide sequences determined herein may contain some error. The automatically determined nucleotide sequence is typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be determined more accurately by other approaches, including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a nucleotide sequence determined in comparison to the actual sequence causes a frame shift in translation of such nucleotide sequence such that the expected amino acid sequence encoded by the determined nucleotide sequence is It will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule beginning at the insertion or deletion point.

Polynucleotides encoding polypeptides of the invention can be obtained from early stage human embryonic (8-9 week) osteoclasts, adult heart or some breast cancer cell lines. The polynucleotides of the present invention are found in cDNA libraries derived from early stage human embryos of ninth week. It is structurally related to the VEGF / PDGF family. It contains an open reading frame that encodes a protein of about 419 amino acid residues, wherein the first approximately 23 amino acid residues of the amino acid residues are putative leader sequences, so the mature protein contains 396 amino acids, and the protein is human vascular endothelial. It shows the highest amino acid sequence homology with the growth factor (30% identity) followed by PDGFa (24%) and PDGFb (22%) (see FIG. 4). Of particular importance is that all eight cysteines are conserved in all four members of the family (boxed in FIG. 3). In addition, PXCVXXXRCXGCCN (SEQ ID NO: 8), which is an indication for the PDGF / VEGF family, is preserved in VEGF-2 (see FIG. 3). Homology between VEGF-2, VEGF and two PDGF is at the protein sequence level. Since no nucleotide sequence homology can be detected, it will be difficult to isolate VEGF-2 through simple approaches such as low stringency hybridization.

VEGF-2 polypeptides of the invention include full length polypeptides and polynucleotide sequences that encode all leader sequences and active fragments of full length polypeptides. The active fragment has less than the full 419 amino acids of the full length amino acid sequence as shown in SEQ ID NO: 2, but the full length amino acid sequence contains 8 cysteine residues shown as conserved in FIG. 3 and still has VEGF-2 activity. Includes all parts of

There are two or more differently spliced VEGF-2 mRNA sequences present in normal tissue. The two bands in FIG. 7, lane 5, indicate the presence of another spliced mRNA encoding the VEGF-2 polypeptide of the invention.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, including cDNA, genomic DNA and synthetic DNA. Such DNA may be double stranded or single stranded. In the case of a Japanese chain, it may be an encrypted chain or a non-encrypted chain (antisense chain). The coding sequence encoding the mature polypeptide may be identical to the coding sequence set forth in FIG. 1 or 2, or the coding sequence of a clone deposited as above, or as a result of overlapping or denaturation of genetic code. Or a different coding sequence that encodes the same mature polypeptide as the DNA of the deposited cDNA.

Polynucleotides encoding the mature polypeptide of FIG. 1 or 2, or a mature polypeptide encoded by a deposited cDNA, include only the coding sequence for the mature polypeptide; Coding sequences for mature polypeptides and additional coding sequences such as liter or secretory sequences or pro-protein sequences; Coding sequences for mature polypeptides (and any additional coding sequences) and non-coding sequences, such as non-coding sequences 5 'and / or 3' or introns of coding sequences for the mature polypeptides may be included. .

Thus, the term "polynucleotide encoding a polypeptide" encompasses polynucleotides comprising only coding sequences for said polypeptides, as well as polynucleotides comprising additional coding and / or non-coding sequences.

The invention further relates to variants of the above-mentioned polynucleotides encoding fragments, homologs and derivatives of the polypeptide having the inferred amino acid sequence of FIG. 1 or 2, or the polypeptide encoded by the cDNA of the deposited clone. Variants of such polynucleotides may be natural allelic variants of the polynucleotides or non-natural variants of the polynucleotides.

Thus, the invention encodes not only a polynucleotide encoding the same mature polypeptide or the same mature polypeptide as deposited in cDNA as shown in Figures 1 or 2, but also the cDNA of the polypeptide or deposited clone of Figure 1 or 2 Variants of such polynucleotides that encode fragments, derivatives or homologs of the polypeptide. Such nucleotide variants include deletion variants, substitution variants, and addition or insertion variants.

As described above, the polynucleotide may have a coding sequence that is a natural allelic variant of the coding sequence set forth in FIG. 1 or 2 or the coding sequence of the deposited clone. As is known in the art, allelic variants are another form of polynucleotide sequence with substitutions, deletions or additions of one or more nucleotides that do not substantially modify the function of the encoded polypeptide.

The present invention also relates to a polynucleotide that aids in the expression and secretion of a polypeptide from a particular host cell, eg, a leader sequence that acts as a secretion sequence to regulate the transport of the polypeptide from the cell. Can be fused within the same read frame as. A polypeptide having a leader sequence is a pre-protein and has a leader sequence that is cleaved by a host cell to form a mature form of polypeptide. The polynucleotide may also encode a pro-protein which is a mature protein plus an additional 5 'amino acid residue. Mature proteins with pro-sequences are pro-proteins, which are proteins in inactive form. When the pro-sequence is cleaved off, the active mature protein remains.

Thus, for example, the polynucleotide of the present invention may encode a mature protein, or a protein having a pro-sequence, or a protein having both a pro-sequence and a pre-sequence (leader sequence).

The polynucleotides of the invention may also have a coding sequence fused in the same frame as the marker sequence that allows purification of the polypeptides of the invention. Such marker sequence may be a hexa-histidine tag supplied by the pQE-9 vector to provide purification of a mature polypeptide fused to the marker in the case of a bacterial host, or for example, the marker sequence may be a mammalian cell, eg For example, when COS-7 cells are used, they may be hemagglutinin (HA) tags. This HA tag corresponds to epitopes derived from influenza hemagglutinin proteins (Wilson, I., et al., Cell 37: 767 (1984)).

A further aspect of the present invention provides a kit comprising: (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence of SEQ ID NO: 2; (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, excluding the N-terminal methionine; (c) a nucleotide sequence encoding a polypeptide having an amino acid sequence from about 1 to 396 of SEQ ID NO: 2; (d) a nucleotide sequence encoding a polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97149; (e) a nucleotide sequence encoding a mature VEGF-2 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97149; Or (f) at least 95%, more preferably 96%, 97%, 98 with the nucleotide sequence complementary to any nucleotide sequence in (a), (b), (c), (d) or (e) above An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence exhibiting at least% or 99% identity.

Further aspects of the invention include (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4; (b) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4, excluding the N-terminal methionine; (c) a nucleotide sequence encoding a polypeptide having an amino acid sequence from about 1 to 326 of SEQ ID NO: 4; (d) a nucleotide sequence encoding a polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 75698; (e) a nucleotide sequence encoding a mature VEGF-2 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 75698; Or (f) at least 95%, more preferably 96%, 97%, 98 with the nucleotide sequence complementary to any nucleotide sequence in (a), (b), (c), (d) or (e) above An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence exhibiting at least% or 99% identity.

A polynucleotide having a reference nucleotide sequence that encodes a VEGF-2 polypeptide and a nucleotide sequence that exhibits, for example, a "same rate" of at least 95%, means that each of the 100 nucleotide sequences of such polynucleotide sequence encodes a VEGF-2 polypeptide. It means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that it may contain up to 5 point mutations per nucleotide. In other words, to obtain a polynucleotide having a nucleotide sequence that exhibits at least 95% identity with a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or the total in the reference sequence Nucleotide numbers up to 5% of the nucleotides may be inserted into the reference sequence. Mutations in such reference sequences can occur anywhere between the 5N or 3N terminal positions of the reference nucleotide sequence, or between said terminal positions, individually inserted between nucleotides in the reference sequence or in one or more adjacent groups within the reference sequence.

In practical terms, a particular nucleic acid molecule exhibits at least 95%, 96%, 97%, 98%, or 99% identity with, for example, the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or the nucleotide sequence of a deposited cDNA clone. Whether or not it is conveniently determined using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) Can be. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981) to find the most similar fragments between the two sequences. When using best bit or other sequence alignment programs to determine whether a particular sequence exhibits 95% identity with, for example, a reference sequence according to the present invention, of course, the same rate is calculated over the entire length of the reference nucleotide sequence. And homology gaps within 5% of the total number of nucleotides in the reference sequence.

As described in detail below, the efficacy of using a polypeptide of the invention for use in diagnostic assays for detecting VEGF-2 protein expression as described below, or to enhance or inhibit VEGF-2 protein function. Polyclonal and monoclonal antibodies useful as agents and antagonists can be produced. In addition, such polypeptides can be used in yeast two-hybrid systems to "capture" VEGF-2 protein binding proteins, which are also candidate agonists and antagonists according to the present invention. Such yeast two-hybrid systems are described in Fields and Song, Nature 340: 245-246 (1989).

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. Regarding the selection of peptides or polypeptides bearing antigenic epitopes (ie, containing specific regions of protein molecules to which antibodies can be bound), relatively short synthetic peptides that mimic part of the protein sequence are typically described above. It is known in the art to be able to induce antisera that react with partially simulated proteins. Sutcliffe, JG, Shinnick, TM, Green, N. and Learner, RA (1983). Peptides capable of inducing protein-reactive serum often appear in the primary sequence of a particular protein and can be characterized by a series of simple chemical rules, which indicate the immunodominant region of the original protein (ie, immunogenic epitope). It is not defined as nor is it defined as amino or carboxy terminus. Extremely hydrophobic peptides and peptides of up to 6 residues are generally ineffective in inducing antibodies that bind to the simulated proteins; Longer soluble peptides, especially those containing proline residues, are commonly effective (Sutcliffe et al., Supra 661). For example, 18 of the 20 peptides designed according to these guidelines, containing 8 to 39 residues, which occupy 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, induce antibodies that react with the HA1 protein or native virus. It was done; 12/12 peptides from MuLV polymerase and 18/18 peptides from ravi glycoproteins induced antibodies that precipitated each protein.

Thus, the antigenic epitope-bearing peptides and polypeptides of the invention are useful for producing antibodies, including monoclonal antibodies, that specifically bind to the polypeptides of the invention. Thus, high proportions of hybridomas obtained by fusion of spleen cells from a donor immunized with an antigenic epitope epitope-bearing peptide generally secrete antibodies that are reactive with the original protein. Sutcliffe et al., 663, supra. Antibodies produced by antigenic epitope-bearing peptides or polypeptides are useful for detecting simulated proteins and antibodies to different peptides can be used to track the trajectories of various regions of the protein precursor undergoing post-detox processing. . Such peptide and anti-peptide antibodies can be used in various qualitative or quantitative assays, eg, competitive assays, for simulated proteins, which can even bind short peptides (eg, about 9 amino acids) and This is because immunoprecipitation assays have been shown to replace larger peptides (Wild, et al., Cell 37: 767-778 (1984) 777). Anti-peptide antibodies of the invention are also useful for purifying proteins simulated by adsorption chromatography, for example using methods well known in the art.

The antigenic epitope-bearing peptides and polypeptides of the invention devised according to the above guidelines are preferably at least 7, more preferably at least 9, even more preferably about 15 to contained within the amino acid sequence of the polypeptide of the invention. It contains at least about 30 amino acids. However, any portion including a larger portion of the amino acid sequence of a polypeptide of the present invention containing about 30, 40, 50, 60, 70, 80, 90, 100 or 150 amino acids, or the entire amino acid sequence of a polypeptide of the present invention. Peptides or polypeptides comprising length are also considered epitope-bearing peptides or polypeptides of the invention and are also useful for inducing antibodies that react with simulated proteins. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to be substantially dissolved in an aqueous solvent (ie, the sequence comprises relatively hydrophilic residues, preferably highly hydrophilic residues); Particularly preferred are sequences containing proline residues.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-2-specific antibodies include polypeptides comprising amino acid residues from about leu-37 to about glu-45 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Tyr-58 to about Gly-66 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Gln-73 to about Glu-81 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Asp-100 to about Cys-108 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Gly-140 to about Leu-148 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Pro-168 to about Val-176 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about His-183 to about Lys-191 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Ile-201 to about Thr-209 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Ala-216 to about Tyr-224 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Asp-244 to about His-254 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Gly-258 to about Glu-266 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Cys-272 to about Ser-280 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Pro-283 to about Ser-291 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Cys-296 to about Gln-304 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Ala-307 to about Cys-316 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Val-319 to about Cys-335 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Cys-339 to about Leu-347 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Cys-360 to about Glu-373 in SEQ ID NO: 2; A polypeptide comprising the amino acid residue of about Tyr-378 to about Val-386 in SEQ ID NO: 2; And a amino acid residue from about Ser-388 to about Ser-396 in SEQ ID NO: 2. These polypeptide fragments were determined to carry the antigenic epitope of the VEGF-2 protein by analysis of the Jamison-Wolf antigen index.

Epitope-bearing peptides and polypeptides of the invention can be prepared by any conventional means for preparing peptides or polypeptides, including recombinant means using the nucleic acid molecules of the invention. For example, short epitope-bearing amino acid sequences can be fused with larger polypeptides that act as carriers during recombinant generation and purification as well as during immunization to generate anti-peptide antibodies. Epitope-bearing peptides can also be synthesized using known chemical synthesis methods. For example, Houghten is a multi-peptide, such as 10-20 mg of 248 different 13 residue peptides that are produced within less than 4 weeks and represent a single amino acid variant of a segment of HA1 polypeptide that has been identified and characterized (by ELISA-type binding studies). A method of simply synthesizing is described. Houghten, RA (1985): General method for rapid solid phase synthesis of multiple peptides: specificity of antigen-antibody interaction at the individual amino acid level. Proc. Natl. Acad. Sci. USA 82: 5131-5135. This “simultaneous multiple peptide synthesis (SMPS)” method is further described in US Pat. No. 4,631,211 (Houghten et al. 1986). In this process, the individual resins for solid phase synthesis of the various peptides are contained in separate solvent-permeable packets that allow optimal use of many of the same repeating steps included in the solid phase process. Due to the complete manual process, 500 to 1000 or more can be synthesized simultaneously (Houghten et al., Supra 5134).

The epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See Sutcliffe et al., Supra; Wilson et al., Supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, F.J. et al., J. Gen. Virol. 66: 2347-2354 (1985). In general, animals can be immunized with free peptides, but anti-peptide antibody titers can be enhanced by coupling the peptides to macromolecular carriers such as keyhole limpet hemacyanine (KLH) or tetanus toxin. For example, cysteine containing peptides can be coupled to the carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides are more common such as glutaraldehyde. Coupling agents may be used to couple to the carrier. Animals such as rabbits, rats, and mice use free peptides or carrier-coupled carriers, for example, by intraperitoneal and / or intradermal injection of an emulsion containing about 100 mg peptide or carrier peptide and Freund's auxiliary solution. Immunize For example, several booster injections may be needed, eg, at about two week intervals, to provide useful titers of anti-peptide antibodies that can be detected by ELISA assays using free peptides adsorbed on solid surfaces. have. The titer of anti-peptide antibodies in serum from immunized animals is increased by selection of anti-peptide antibodies, for example by adsorbing to peptides on solid supports and eluting the selected antibodies according to methods well known in the art. You can.

The immunogenic epitope-bearing peptides of the invention, ie, the parts of the protein which elicit an antibody response when the whole protein is an immunogen, are identified according to methods known in the art. Geysen et al., Supra, describe methods for the rapid and simultaneous synthesis of numerous peptides on a solid support that are sufficiently pure to react in enzyme-linked immunosorbent assays. The interaction between the synthesized peptide and the antibody is then easily detected without removing the support from them. In this way, peptides bearing immunogenic epitopes of the protein of interest can be routinely identified by those skilled in the art. For example, according to the literature, an immunologically important epitope in the coat protein of the foot and oral disease virus is 7 by synthesizing an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Localized with the splitting of amino acids. Subsequently, a complete replacement set of peptides is substituted, in which all 20 amino acids are substituted at all positions within the epidope, to determine specific amino acids that confer specificity for reaction with the antibody. Thus, peptide homologs of epitope-bearing peptides of the invention can be conventionally prepared in this manner. U.S. Patent 4,708,781 (Geysen; 1987) describes the above method of identifying peptides that bear immunogenic epitopes of the protein of interest.

In addition, US Pat. No. 5,194,392 (Geysen, 1990) discloses monomers that are morphological equivalents of epitopes (ie, “Aminotope”) that are complementary to a specific paratope (antigen binding site) of an antibody of interest. General methods for detecting or determining sequences of (amino acids or other compounds) are described. More generally, US Pat. No. 4,433,092 (Geysen, 1989) describes methods for detecting or determining the sequence of monomers, which are morphological equivalents of ligands complementary to the ligand binding site of a particular receptor of interest. Similarly, US Pat. No. 5,480,971 (Houghten, RA et al., 1960) on peralkylated oligopeptide mixtures discloses C ₁ -C ₇ alkyl peralkylated oligopeptides and sets and libraries of such peptides as well as receptor molecules of interest. Methods of using such oligopeptide sets and libraries to determine the sequence of peralkylated oligopeptides that are preferentially bound are described. Thus, non-peptide homologs of epitope-bearing peptides of the present invention can be prepared conventionally by these methods.

Those skilled in the art will appreciate that the VEGF-2 polypeptides of the invention and epitope-bearing fragments thereof as mentioned above can be combined with a portion of the constant region of an immunoglobulin (IgG) to produce chimeric polypeptides. These fusion proteins promote purification and increase half-life in vivo. This has been shown, for example, for chimeric proteins consisting of various regions of the first two regions of human CD4-polypeptide and the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP A 394,827; Traunecker et al., Nature 331: 84-86 (1988).

According to the present invention, novel variants of VEGF-2 have been described. These can be produced by deleting or replacing one or more amino acids of VEGF-2. Natural mutations are called allelic variations. Allelic variation may be asymptomatic (no change in the encoded polypeptide) or may have a modified amino acid sequence.

In order to improve or modify the characteristics of the original VEGF-2, protein engineering can be used. Recombinant DNA techniques known to those skilled in the art can be used to generate novel polypeptides. Muteins and deletions may exhibit, for example, enhanced activity or increased stability. In addition, they can be purified in high yields and exhibit better solubility, at least under certain purification and storage conditions. Examples of mutants that can be constructed are shown below.

Amino and carboxy terminal deletions

In addition, VEGF-2 is proteolytically cleaved upon expression and is believed to produce polypeptide fragments of the following sizes when performed on an SDS-PAGE gel (size is approximate) (see, eg, FIGS. 6-8). ): 80, 59, 45, 43, 41, 40, 39, 38, 37, 36, 31, 29, 21 and 15 kDa. These polypeptide fragments are proteolytic cleavage products at both the N-terminal and C-terminal sites of the protein. These proteolytically generated fragments, in particular 21 kDa fragments, are believed to be active.

In addition, protein engineering can be used to ameliorate or modify the characteristics of one or more of the original VEGF-2s. Deletion of carboxy terminal amino acids can enhance protein activity. One example is interferon gamma, which exhibits a 10-fold higher activity by deleting 10 amino acid residues from the carboxy terminus of a protein (Dobeli et al., J. of Biotechnology 7: 199-216 (1988)). Thus, one aspect of the invention is polypeptide homologues of VEGF-2 and such homologs that exhibit enhanced stability compared to the original VEGF-2 polypeptide (eg, when exposed to typical pH, thermal conditions or other storage conditions). To provide a nucleotide sequence encoding the.

Particularly preferred VEGF-2 polypeptides are shown below (numbered starting with the first amino acid (Met) in the protein) (FIG. 1 (SEQ ID NO: 18)): Ala (residue 24) to Ser (residue 419); Pro 25 to Ser 419; Ala 26 to Ser 419; Ala 27 to Ser 419; Ala (28) to Ser (419); Ala (29) to Ser (419); Ala 30 to Ser 419; Leu (31) to Ser (419); Glu (32) to Ser (419); Ser 33 to Ser 419; Gly (34) to Ser (419); Lue (35) to Ser (419); Asp 36 to Ser 419; Leu (37) to Ser (419); Ser 38 to Ser 419; Asp 39 to Ser 419; Ala 40 to Ser 419; Glu 41 to Ser 419; Pro 42 to Ser 419; Asp 43 to Ser 419; Ala 44 to Ser 419; Gly (45) to Ser (419); Glu (46) to Ser (419); Ala (47) to Ser (419); Thr (48) to Ser (419); Ala 49 to Ser 419; Tyr (50) to Ser (419); Ser 52 to Ser 419; Asp 54 to Ser 419; Val 62 to Ser 419; Val 65 to Ser 419; Met (1), Glu (23), or Ala (24) to Met (418); Met (1), Glu (23), or Ala (24) to Glu (417); Met (1), Glu (23), or Ala (24) to Pro (416); Met (1), Glu (23), or Ala (24) or Arg (415); Met (1), Glu (23), or Ala (24) to Gln (414); Met (1), Glu (23), or Ala (24) to Trp (413); Met (1), Glu (23), or Ala (24) to Tyr (412); Met (1), Glu (23), or Ala (24) to Ser (411); Met (1), Glu (23), or Ala (24) to Pro (410); Met (1), Glu (23), or Ala (24) to Val (409); Met (1), Glu (23), or Ala (24) to Cys (408); Met (1), Glu (23), or Ala (24) to Arg (407); Met (1), Glu (23), or Ala (24) to Cys (406); Met (1), Glu (23) or Ala (24) to Val (405); Met (1), Glu (23), or Ala (24) to Glu (404); Met (1), Glu (23), or Ala (24) to Glu (403); Met (1), Glu (23), or Ala (24) to Ser (402); Met (1), Glu (23), or Ala (24) to Gly (398); Met (1), Glu (23), or Ala (24) to Pro (397); Met (1), Glu (23), or Ala (24) to Lys (393); Met (1), Glu (23), or Ala (24) to Met (263); Met (1), Glu (23), or Ala (24) to Asp (311); Met (1), Glu (23), or Ala (24) to Pro (367); Met (1) to Ser (419); Met (1) to Ser (228); Glu (47) to Ser (419); Ala (111) to Lys (214); Ala 112 to Lys 214; His (113) to Lys (214); Tyr (114) to Lys (214); Asn 115 to Lys 214; Thr 116 to Lys 214; Thr 103 to Leu 215; Glu 104 to Leu 215; Glu 105 to Leu 215; Thr 106 to Leu 215; Ile 107 to Leu 215; Lys 108 to Leu 215; Phe 109 to Leu 215; Ala 110 to Leu 215; Ala (111) to Leu (215); Ala 112 to Leu 215; His (113) to Leu (215); Tyr 114 to Leu 215; Asn 115 to Leu 215; Thr 116 to Leu 215; Thr 103-Ser 228; Glu 104 to Ser 228; Glu 105 to Ser 228; Thr 106-Ser 228; Ile 107 to Ser 228; Lys 108 to Ser 228; Phe 109 to Ser 228; Ala 110 to Ser 228; Ala 111-Ser 228; Ala 112 to Ser 228; His (113) to Ser (228); Tyr 114 to Ser 228; Asn 115 to Ser 228; Thr 116 to Ser 228; Thr (103) to Leu (229); Glu (104) to Leu (229); Thr 103-Arg 227; glu 104 to Arg 227; Glu 105 to Arg 227; Thr 106-Arg 227; Ile 107 to Arg 227; Lys 108 to Arg 227; Phe 109 to Arg 227; Ala 110 to Arg 227; Ala (111) to Arg (227); Ala 112 to arg 227; His (113) to Arg (227); Tyr 114 to Arg 227; Asn 115 to Arg 227; Thr 116 to Arg 227; Thr 103 to Ser 213; Glu 104 to Ser 213; Glu 105 to Ser 213; Thr 106 to Ser 213; Ile 107 to Ser 213; Lys 108 to Ser 213; Phe 109 to Ser 213; Ala 110 to Ser 213; Ala 111 to Ser 213; Ala 112 to Ser 213; His (113) to Ser (213); Tyr 114 to Ser 213; Asn 115 to Ser 213; Thr 116 to Ser 213; Thr (103) to Lys (214); Glu (104) to Lys (214); Glu 105 to Lys 214; Thr (106) to Lys (214); Ile 107 to Lys 214; Lys 108 to Lys 214; Phe 109 to Lys 214; Ala 110-Lys 214; Glu 105 to Leu 229; Thr (106) to Leu (229); Ile 107 to Leu 229; Lys (108) to Leu (229); Phe 109 to Leu 229; Ala 110 to Leu 229; Ala (111) to Leu (229); Ala 112 to Leu 229; His (113) to Leu (229); Tyr (114) to Leu (229); Asn 115 to Leu 229; Thr (116) to Leu (229).

Preferred embodiments include the following deletion mutants: Thr (103) -Arg (227); Glu (104) -Arg (227); Ala 212-Arg 227; Thr 103-Ser 213; Glu (104) -Ser (213); Thr (103) -Leu (215); Glu (47) -Ser (419); Met (1), Glu (23), or Ala (24) -Met (263); Met (1), Glu (23), or Ala (24) -Asp (311); Met (1), Glu (23), or Ala (24) -Pro (367); Met (1) -Ser (119); And Met (1) -Ser (228) (FIG. 1 (SEQ ID NO: 18)).

Also included in the present invention are deletion mutants having amino acids deleted from both the NB terminus and the C-terminus. Such mutants include all combinations of the above-mentioned N-terminal deletion mutants and C-terminal deletion mutants. These combinations can be made using recombinant techniques known to those skilled in the art.

In particular, the N-terminal deletion of the VEGF-2 polypeptide can be described by the general formula m-396, where m is an integer from -23 to 388 and corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. Preferably, the N-terminal deletion has a conserved boxed moiety (PXCVXXXRCXGCCN) (SEQ ID NO: 8) of FIG. 3 and comprises a polypeptide comprising the amino acid sequence of a residue: the present invention as set forth in SEQ ID NO: 1 N-terminal deletions of the polypeptide of include polypeptides comprising the amino acid sequence of the following residues: E-1 to S-396; A-2 to S-396; P-3 to S-396; A-4 to S-396; A-5 to S-396; A-6 to S-396; A-7 to S-396; A-8 to S-396; F-9 to S-396; E-10 to S-396; S-11 to S-396; G-12 to S-396; L-13 to S-396; D-14 to S-396; L-15 to S-396; S-16 to S-396; D-17 to S-396; A-18 to S-396; E-19 to S-396; P-20 to S-396; D-21 to S-396; A-22 to S-396; G-23 to S-396; E-24 to S-396; A-25 to S-396; T-26 to S-396; A-27 to S-396; Y-28 to S-396; A-29 to S-396; S-30 to S-396; K-31 to S-396; D-32 to S-396; L-33 to S-396; E-34 to S-396; E-35 to S-396; Q-36 to S-396; L-37 to S-396; R-38 to S-396; S-39 to S-396; V-40 to S-396; S-41 to S-396; S-42 to S-396; V-43 to S-396; D-44 to S-396; E-45 to S-396; L-46 to S-396; M-47 to S-396; T-48 to S-396; V-49 to S-396; L-50 to S-396; Y-51 to S-396; P-52 to S-396; E-53 to S-396; Y-54 to S-396; W-55 to S-396; K-56 to S-396; M-57 to S-396; Y-58 to S-396; K-59 to S-396; C-60 to S-396; Q-61 to S-396; L-62 to S-396; R-63 to S-396; K-64 to S-396; G-65 to S-396; G-66 to S-396; W-67 to S-396; Q-68 to S-396; H-69 to S-396; N-70 to S-396; R-71 to S-396; E-72 to S-396; Q-73 to S-396; A-74 to S-396; N-75 to S-396; L-76 to S-396; N-77 to S-396; S-78 to S-396; R-79 to S-396; T-80 to S-396; E-81 to S-396; E-82 to S-396; T-83 to S-396; I-84 to S-396; K-85 to S-396; F-86 to S-396; A-87 to S-396; A-88 to S-396; A-89 to S-396; H-90 to S-396; Y-91 to S-396; N-92 to S-396; T-93 to S-396; E-94 to S-396; I-95 to S-396; K-97 to S-396; K-77 to S-396; S-98 to S-396; I-99 to S-396; D-100 to S-396; N-101 to S-396; E-102 to S-396; W-103 to S-396; R-104 to S-396; K-105 to S-396; T-106 to S-396; Q-107 to S-396; C-108 to S-396; M-109 to S-396; P-110 to S-396; R-111 to S-396; E-112 to S-396; V-113 to S-396; C-114 to S-396; I-115 to S-396; D-116 to S-396; V-117 to S-396; G-118 to S-396; K-119 to S-396; E-120 to S-396; F-121 to S-396; G-122 to S-396; V-123 to S-396; A-124 to S-396; T-125 to S-396; N-126 to S-396; T-127 to S-396; F-128 to S-396; F-129 to S-396; K-130 to S-396; P-131 to S-396; P-132 to S-396; C-133 to S-396; V-134 to S-396; S-135 to S-396; V-136 to S-396; Y-137 to S-396; R-138 to S-396; C-139 to S-396; G-140 to S-396; G-141 to S-396; C-142 to S-396; C-143 to S-396; N-144 to S-396; S-145 to S-396; E-146 to S-396; G-147 to S-396; L-148 to S-396; Q-149 to S-396; C-150 to S-396; M-151 to S-396; N-152 to S-396; T-153 to S-396; S-154 to S-396; T-155 to S-396; S-156 to S-396; Y-157 to S-396; L-158 to S-396; S-159 to S-396; K-160 to S-396; T-161 to S-396; L-162 to S-396; F-163 to S-396; E-164 to S-396; I-165 to S-396; T-166 to S-396; V-167 to S-396; P-168 to S-396; L-169 to S-396; S-170 to S-396; Q-171 to S-396; G-172 to S-396; P-173 to S-396; K-174 to S-396; P-175 to S-396; V-176 to S-396; T-177 to S-396; I-178 to S-396; S-179 to S-396; F-180 to S-396; A-181 to S-396; N-182 to S-396; H-183 to S-396; T-184 to S-396; S-185 to S-396; C-186 to S-396; R-187 to S-396; C-188 to S-396; M-189 to S-396; S-190 to S-396; K-191 to S-396; L-192 to S-396; D-193 to S-396; V-194 to S-396; Y-195 to S-396; R-196 to S-396; Q-197 to S-396; V-198 to S-396; H-199 to S-396; S-200 to S-396; I-201 to S-396; I-202 to S-396; R-203 to S-396; R-204 to S-396; S-205 to S-396; L-206 to S-396; P-207 to S-396; A-208 to S-396; T-209 to S-396; L-210 to S-396; P-211 to S-396; Q-212 to S-396; C-213 to S-396; Q-214 to S-396; A-215 to S-396; A-216 to S-396; N-217 to S-396; K-218 to S-396; T-219 to S-396; C-220 through S-396; P-221 to S-396; T-222 to S-396; N-223 to S-396; Y-224 to S-396; M-225 to S-396; W-226 to S-396; N-227 to S-396; N-228 to S-396; H-229 to S-396; I-230 to S-396; C-231 to S-396; R-232 to S-396; C-233 through S-396; L-234 to S-396; A-235 to S-396; Q-236 to S-396; E-237 to S-396; D-238 to S-396; F-239 to S-396; M-240 to S-396; F-241 to S-396; S-242 to S-396; S-243 to S-396; D-244 to S-396; A-245 to S-396; G-246 to S-396; D-247 to S-396; D-248 to S-396; S-249 to S-396; T-250 to S-396; D-251 to S-396; G-252 to S-396; F-253 to S-396; H-254 to S-396; D-255 to S-396; I-256 to S-396; C-257 to S-396; G-258 to S-396; P-259 to S-396; N-260 to S-396; K-261 to S-396; E-262 to S-396; L-263 to S-396; D-264 to S-396; E-265 to S-396; E-266 to S-396; T-267 to S-396; C-268 to S-396; Q-269 to S-396; C-270 to S-396; V-271 to S-396; C-272 to S-396; R-273 to S-396; A-274 to S-396; G-275 to S-396; L-276 to S-396; R-277 to S-396; P-278 to S-396; A-279 to S-396; S-280 to S-396; C-281 to S-396; G-282 to S-396; P-283 to S-396; H-284 to S-396; K-285 to S-396; E-286 to S-396; L-287 to S-396; D-288 to S-396; R-289 to S-396; N-290 to S-396; S-291 to S-396; C-292 to S-396; Q-293 to S-396; C-284 to S-396; V-295 to S-396; C-296 to S-396; K-297 to S-396; N-298 to S-396; K-299 to S-396; L-300 to S-396; F-301 to S-396; P-302 to S-396; S-303 to S-396; Q-304 to S-396; C-305 to S-396; G-306 to S-396; A-307 to S-396; N-308 to S-396; R-309 to S-396; E-310 to S-396; F-311 to S-396; D-312 to S-396; E-313 to S-396; N-314 to S-396; T-315 to S-396; C-316 to S-396; Q-317 to S-396; C-318 to S-396; V-319 to S-396; C-320 to S-396; K-321 to S-396; R-322 to S-396; T-323 to S-396; C-324 to S-396; P-325 to S-396; R-326 to S-396; N-327 to S-396; Q-328 to S-396; P-329 to S-396; L-330 to S-396; N-331 to S-396; P-332 to S-396; G-333 to S-396; K-334 to S-396; C-335 through S-396; A-336 to S-396; C-337 to S-396; E-338 to S-396; C-339 to S-396; T-340 to S-396; E-341 to S-396; S-342 to S-396; P-343 to S-396; Q-344 to S-396; K-345 to S-396; C-346 to S-396; L-347 to S-396; L-348 to S-396; K-349 to S-396; G-350 to S-396; K-351 to S-396; K-352 to S-396; F-353 to S-396; H-354 to S-396; H-355 to S-396; Q-356 to S-396; T-357 to S-396; C-358 to S-396; S-359 to S-396; C-360 to S-396; Y-361 to S-396; R-362 to S-396; R-363 to S-396; P-364 to S-396; C-365 to S-396; T-366 to S-396; N-367 to S-396; R-368 to S-396; Q-369 to S-396; K-370 to S-396; A-371 to S-396; C-372 to S-396; E-373 to S-396; P-374 to S-396; G-375 to S-396; F-376 to S-396; S-377 to S-396; Y-378 to S-396; S-379 to S-396; E-380 through S-396; E-381 to S-396; V-382 to S-396; C-383 through S-396; R-384 to S-396; C-385 to S-396; V-386 to S-396; P-387 to S-396; S-388 to S-396; Y-389 to S-396; W-390 to S-396; Q-391 to S-396 [SEQ ID NO: 2]. One preferred embodiment comprises amino acids S-205 to S-396 of SEQ ID NO: 2. Also preferred are polynucleotides encoding these polypeptides.

Moreover, the C-terminal deletion of the VEGF-2 polypeptide can be described by the general formula -23-n, where n is an integer from -15 to 395 and corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. . Preferably, the C-terminal deletion has a conserved boxed moiety (PXCVXXXRCXGCCN) of SEQ ID NO. 3 (SEQ ID NO: 8) and comprises a polypeptide comprising the amino acid sequence of the residue: likewise as shown in SEQ ID NO: 2 C-terminal deletions of the polypeptides of the invention include polypeptides comprising the amino acid sequence of the following residues: E-1 to M-395; E-1 to Q-394; E-1 to P-393; E-1 to R-392; E-1 to Q-391; E-1 to W-390; E-1 to Y-389; E-1 to S-388; E-1 to P-387; E-1 to V-386; E-1 to C-385; E-1 to R-384; E-1 to C-383; E-1 to V-382; E-1 to E-381; E-1 to E-380; E-1 to S-379; E-1 to Y-378; E-1 to S-377; E-1 to F-376; E-1 to G-375; E-1 to P-374; E-1 to E-373; E-1 to C-372; E-1 to A-371; E-1 to K-370; E-1 to Q-369; E-1 to R-368; E-1 to N-367; E-1 to T-366; E-1 to C-365; E-1 to P-364; E-1 to R-363; E-1 to R-362; E-1 to Y-361; E-1 to C-360; E-1 to S-359; E-1 to C-358; E-1 to T-357; E-1 to Q-356; E-1 to H-355; E-1 to H-354; E-1 to F-353; E-1 to K-352; E-1 to K-351; E-1 to G-350; E-1 to K-349; E-1 to L-348; E-1 to L-347; E-1 to C-346; E-1 to K-345; E-1 to Q-344; E-1 to P-343; E-1 to S-342; E-1 to E-341; E-1 to T-340; E-1 to C-339 E-1 to E-338; E-1 to C-337; E-1 to A-336; E-1 to C-335; E-1 to K-334; E-1 to G-333; E-1 to P-332; E-1 to N-331; E-1 to L-330; E-1 to P-329; E-1 to Q-328; E-1 to N-327; E-1 to R-326; E-1 to P-325; E-1 to C-324; E-1 to T-323; E-1 to R-322; E-1 to K-321; E-1 to C-320; E-1 to V-319; E-1 to C-318; E-1 to Q-317; E-1 to C-316; E-1 to T-315; E-1 to N-314; E-1 to E-313; E-1 to D-312; E-1 to F-311; E-1 to E-310; E-1 to R-309; E-1 to N-308; E-1 to A-307; E-1 to G-306; E-1 to C-305; E-1 to Q-304; E-1 to S-303; E-1 to P-302; E-1 to F-301; E-1 to L-300; E-1 to K-299; E-1 to N-298; E-1 to K-297; E-1 to C-296; E-1 to V-295; E-1 to C-294; E-1 to Q-293; E-1 to C-292; E-1 to S-291; E-1 to N-290; E-1 to R-289; E-1 to D-288; E-1 to L-287; E-1 to E-286; E-1 to K-285; E-1 to H-284; E-1 to P-283; E-1 to G-282; E-1 to C-281; E-1 to S-280; E-1 to A-279; E-1 to P-278; E-1 to R-277; E-1 to L-276; E-1 to G-275; E-1 to A-274; E-1 to R-273; E-1 to C-272; E-1 to V-271; E-1 to C-270; E-1 to Q-269; E-1 to C-268; E-1 to T-267; E-1 to E-266; E-1 to E-265; E-1 to D-264; E-1 to L-263; E-1 to E-262; E-1 to K-261; E-1 to N-260; E-1 to P-259; E-1 to G-258; E-1 to C-257; E-1 to I-256; E-1 to D-255; E-1 to H-254; E-1 to F-253; E-1 to G-252; E-1 to D-251; E-1 to T-250; E-1 to S-249; E-1 to D-248; E-1 to D-247; E-1 to G-246; E-1 to A-245; E-1 to D-244; E-1 to S-243; E-1 to S-242; E-1 to F-241; E-1 to M-240; E-1 to F-239; E-1 to D-238; E-1 to E-237; E-1 to Q-236; E-1 to A-235; E-1 to L-234; E-1 to C-233; E-1 to R-232; E-1 to C-231; E-1 to I-230; E-1 to H-229; E-1 to N-228; E-1 to N-227; E-1 to W-226; E-1 to M-225; E-1 to Y-224; E-1 to N-223; E-1 to T-222; E-1 to P-221; E-1 to C-220; E-1 to T-219; E-1 to K-218; E-1 to N-217; E-1 to A-216; E-1 to A-215; E-1 to Q-214; E-1 to C-213; E-1 to Q-212; E-1 to P-211; E-1 to L-210; E-1 to T-209; E-1 to A-208; E-1 to P-207; E-1 to L-206; E-1 to S-205; E-1 to R-204; E-1 to R-203; E-1 to I-202; E-1 to I-201; E-1 to S-200; E-1 to H-199; E-1 to V-198; E-1 to Q-197; E-1 to R-196; E-1 to Y-195; E-1 to V-194; E-1 to D-193; E-1 to L-192; E-1 to K-191; E-1 to S-190; E-1 to M-189; E-1 to C-188; E-1 to R-187; E-1 to C-186; E-1 to S-185; E-1 to T-184; E-1 to H-183; E-1 to N-182; E-1 to A-181; E-1 to F-180; E-1 to S-179; E-1 to I-178; E-1 to T-177; E-1 to V-176; E-1 to P-175; E-1 to K-®; E-1 to P-173; E-1 to G-172; E-1 to Q-171; E-1 to S-170; E-1 to L-169; E-1 to P-168; E-1 to V-167; E-1 to T-166; E-1 to I-165; E-1 to E-164; E-1 to F-163; E-1 to L-162; E-1 to T-161; E-1 to K-160; E-1 to S-159; E-1 to L-158; E-1 to Y-157; E-1 to S-156; E-1 to T-155; E-1 to S-154; E-1 to T-153; E-1 to N-152; E-1 to M-151; E-1 to C-150; E-1 to Q-149; E-1 to L-148; E-1 to G-147; E-1 to E-146; E-1 to S-145; E-1 to N-144; E-1 to C-143; E-1 to C-142; E-1 to G-141; E-1 to G-140; E-1 to C-139; E-1 to R-138; E-1 to Y-137; E-1 to V-136; E-1 to S-135; E-1 to V-134; E-1 to C-133; E-1 to P-132; E-1 to P-131; E-1 to K-130; E-1 to F-129; E-1 to F-128; E-1 to T-127; E-1 to N-126; E-1 to T-125; E-1 to A-124; E-1 to V-123; E-1 to G-122; E-1 to F-121; E-1 to E-120; E-1 to K-119; E-1 to G-118; E-1 to V-117; E-1 to D-116; E-1 to I-115; E-1 to C-114; E-1 to V-113; E-1 to E-112; E-1 to R-111; E-1 to P-110; E-1 to M-109; E-1 to C-108; E-1 to Q-107; E-1 to T-106; E-1 to K-105; E-1 to R-104; E-1 to W-103; E-1 to E-102; E-1 to N-101; E-1 to D-100; E-1 to I-99; E-1 to S-98; E-1 to K-97; E-1 to L-96; E-1 to I-95; E-1 to E-94; E-1 to T-93; E-1 to N-92; E-1 to Y-91; E-1 to H-90; E-1 to A-89; E-1 to A-88; E-1 to A-87; E-1 to F-86; E-1 to K-85; E-1 to I-84; E-1 to T-83; E-1 to E-82; E-1 to E-81; E-1 to T-80; E-1 to R-79; E-1 to S-78; E-1 to N-77; E-1 to L-76; E-1 to N-75; E-1 to A-74; E-1 to Q-73; E-1 to E-72; E-1 to R-71; E-1 to N-70; E-1 to H-69; E-1 to Q-68; E-1 to W-67; E-1 to G-66; E-1 to G-65; E-1 to K-64; E-1 to R-63; E-1 to L-62; E-1 to Q-61; E-1 to C-60; E-1 to K-59; E-1 to Y-58; E-1 to M-57; E-1 to K-56; E-1 to W-55; E-1 to Y-54; E-1 to E-53; E-1 to P-52; E-1 to Y-51; E-1 to L-50; E-1 to V-49; E-1 to T-48; E-1 to M-47; E-1 to L-46; E-1 to E-45; E-1 to D-44; E-1 to V-43; E-1 to S-42; E-1 to S-41; E-1 to V-40; E-1 to S-39; E-1 to R-38; E-1 to L-37; E-1 to Q-36; E-1 to E-35; E-1 to E-34; E-1 to L-33; E-1 to D-32; E-1 to K-31; E-1 to S-30; E-1 to A-29; E-1 to Y-28; E-1 to A-27; E-1 to T-26; E-1 to A-25; E-1 to E-24; E-1 to G-23; E-1 to A-22; E-1 to D-21; E-1 to P-20; E-1 to E-19; E-1 to A-18; E-1 to D-17; E-1 to S-16; E-1 to L-15; E-1 to D-14; E-1 to L-13; E-1 to G-12; E-1 to S-11; E-1 to E-10; E-1 to F-9; E-1 to A-8; E-1 to A-7 (SEQ ID NO: 2). Also preferred are polynucleotides encoding these polypeptides.

Moreover, the present invention also relates to one or more amino acids deleted from both the amino and carboxy termini, which may be generally described as having residues mn of SEQ ID NO: 2, wherein n and m are integers as mentioned above. It provides a polypeptide having.

Likewise preferred are C-terminal deletions of the VEGF-2 polypeptides of the invention as set forth in SEQ ID NO: 2, comprising a polypeptide comprising an amino acid sequence of the following residues: F-9 to M-395; F-9 to Q-394; F-9 to P-393; F-9 to R-392; F-9 to Q-391; F-9 to W-390; F-9 to Y-389; F-9 to S-388; F-9 to P-387; F-9 to V-386; F-9 to C-385; F-9 to R-384; F-9 to C-383; F-9 to V-382; F-9 to E-381; F-9 to E-380; F-9 to S-379; F-9 to Y-378; F-9 to S-377; F-9 to F-376; F-9 to G-375; F-9 to P-374; F-9 to E-373; F-9 to C-372; F-9 to A-371; F-9 to K-370; F-9 to Q-369; F-9 to R-368; F-9 to R-367; F-9 to T-366; F-9 to C-365; F-9 to P-364; F-9 to R-363; F-9 to R-362; F-9 to Y-361; F-9 to C-360; F-9 to S-359; F-9 to C-358; F-9 to T-357; F-9 to Q-356; F-9 to H-355; F-9 to H-354; F-9 to F-353; F-9 to K-352; F-9 to K-351; F-9 to G-350; F-9 to K-349; F-9 to L-348; F-9 to L-347; F-9 to C-346; F-9 to K-345; F-9 to Q-344; F-9 to P-343; F-9 to S-342; F-9 to E-341; F-9 to T-340; F-9 to C-339; F-9 to E-338; F-9 to C-337; F-9 to A-336; F-9 to C-335; F-9 to K-334; F-9 to G-333; F-9 to P-332; F-9 to N-331; F-9 to L-330; F-9 to P-329; F-9 to Q-328; F-9 to N-327; F-9 to R-326; F-9 to P-325; F-9 to C-324; F-9 to T-323; F-9 to R-322; F-9 to K-321; F-9 to C-320; F-9 to V-319; F-9 to C-318; F-9 to Q-317; F-9 to C-316; F-9 to T-315; F-9 to N-314; F-9 to E-313; F-9 to D-312; F-9 to F-311; F-9 to E-310; F-9 to R-309; F-9 to N-308; F-9 to A-307; F-9 to G-306; F-9 to C-305; F-9 to Q-304; F-9 to S-303; F-9 to P-302; F-9 to F-301; F-9 to L-300; F-9 to k-299; F-9 to N-298; F-9 to K-297; F-9 to C-296; F-9 to V-295; F-9 to C-294; F-9 to Q-293; F-9 to C-292; F-9 to S-291; F-9 to N-290; F-9 to R-289; F-9 to D-288; F-9 to L-287; F-9 to E-286; F-9 to K-285; F-9 to H-284; F-9 to P-283; F-9 to G-282; F-9 to C-281; F-9 to S-280; F-9 to A-279; F-9 to P-278; F-9 to R-277; F-9 to L-276; F-9 to G-275; F-9 to A-274; F-9 to R-273; F-9 to C-272; F-9 to V-271; F-9 to C-270; F-9 to Q-269; F-9 to C-268; F-9 to T-267; F-9 to E-266; F-9 to E-265; F-9 to D-264; F-9 to L-263; F-9 to E-262; F-9 to K-261; F-9 to N-260; F-9 to P-259; F-9 to G-258; F-9 to C-257; F-9 to I-256; F-9 to D-255; F-9 to H-254; F-9 to F-253; F-9 to G-252; F-9 to D-251; F-9 to T-250; F-9 to S-249; F-9 to D-248; F-9 to D-247; F-9 to G-246; F-9 to A-245; F-9 to D-244; F-9 to S-243; F-9 to S-242; F-9 to F-241; F-9 to M-240; F-9 to F-239; F-9 to D-238; F-9 to E-237; F-9 to Q-236; F-9 to A-235; F-9 to L-234; F-9 to C-233; F-9 to R-232; F-9 to C-231; F-9 to I-230; F-9 to H-229; F-9 to N-228; F-9 to N-227; F-9 to W-226; F-9 to M-225; F-9 to Y-224; F-9 to N-223; F-9 to T-222; F-9 to P-221; F-9 to C-220; F-9 to T-219; F-9 to K-218; F-9 to N-217; F-9 to A-216; F-9 to A-215; F-9 to Q-214; F-9 to C-213; F-9 to Q-212; F-9 to P-211; F-9 to L-210; F-9 to T-209; F-9 to A-208; F-9 to P-207; F-9 to L-206; F-9 to S-205; F-9 to R-204; F-9 to R-203; F-9 to I-202; F-9 to I-201; F-9 to S-200; F-9 to H-199; F-9 to V-198; F-9 to Q-197; F-9 to R-196; F-9 to Y-195; F-9 to V-194; F-9 to D-193; F-9 to L-192; F-9 to K-191; F-9 to S-190; F-9 to M-189; F-9 to C-188; F-9 to R-187; F-9 to C-186; F-9 to S-185; F-9 to T-184; F-9 to H-183; F-9 to N-182; F-9 to A-181; F-9 to F-180; F-9 to S-179; F-9 to I-178; F-9 to T-177; F-9 to V-176; F-9 to P-175; F-9 to K-®; F-9 to P-173; F-9 to G-172; F-9 to Q-171; F-9 to S-170; F-9 to L-169; F-9 to P-168; F-9 to V-167; F-9 to T-166; F-9 to I-165; F-9 to E-164; F-9 to F-163; F-9 to L-162; F-9 to T-161; F-9 to K-160; F-9 to S-159; F-9 to L-158; F-9 to Y-157; F-9 to S-156; F-9 to T-155; F-9 to S-154; F-9 to T-153; F-9 to N-152; F-9 to M-151; F-9 to C-150; F-9 to Q-149; F-9 to L-148; F-9 to G-147; F-9 to E-146; F-9 to S-145; F-9 to N-144; F-9 to C-143; F-9 to C-142; F-9 to G-141; F-9 to G-140; F-9 to C-139; F-9 to R-138; F-9 to Y-137; F-9 to V-136; F-9 to S-135; F-9 to V-134; F-9 to C-133; F-9 to P-132; F-9 to P-131; F-9 to K-130; F-9 to F-129; F-9 to F-128; F-9 to T-127; F-9 to N-126; F-9 to T-125; F-9 to A-124; F-9 to V-123; F-9 to G-122; F-9 to F-121; F-9 to E-120; F-9 to K-119; F-9 to G-118; F-9 to V-117; F-9 to D-116; F-9 to I-115; F-9 to C-114; F-9 to V-113; F-9 to E-112; F-9 to R-111; F-9 to P-110; F-9 to M-109; F-9 to C-108; F-9 to Q-107; F-9 to T-106; F-9 to K-105; F-9 to R-104; F-9 to W-103; F-9 to E-102; F-9 to N-101; F-9 to D-100; F-9 to I-99; F-9 to S-98; F-9 to K-97; F-9 to L-96; F-9 to I-95; F-9 to E-94; F-9 to T-93; F-9 to N-92; F-9 to Y-91; F-9 to H-90; F-9 to A-89; F-9 to A-88; F-9 to A-87; F-9 to F-86; F-9 to K-85; F-9 to I-84; F-9 to T-83; F-9 to E-82; F-9 to E-81; F-9 to T-80; F-9 to R-79; F-9 to S-78; F-9 to N-77; F-9 to L-76; F-9 to N-75; F-9 to A-74; F-9 to Q-73; F-9 to E-72; F-9 to R-71; F-9 to N-70; F-9 to H-69; F-9 to Q-68; F-9 to W-67; F-9 to G-66; F-9 to G-65; F-9 to K-64; F-9 to R-63; F-9 to L-62; F-9 to Q-61; F-9 to C-60; F-9 to K-59; F-9 to Y-58; F-9 to M-57; F-9 to K-56; F-9 to W-55; F-9 to Y-54; F-9 to E-53; F-9 to P-52; F-9 to Y-51; F-9 to L-50; F-9 to V-49; F-9 to T-48; F-9 to M-47; F-9 to L-46; F-9 to E-45; F-9 to D-44; F-9 to V-43; F-9 to S-42; F-9 to S-41; F-9 to V-40; F-9 to S-39; F-9 to R-38; F-9 to L-37; F-9 to Q-36; F-9 to E-35; F-9 to E-34; F-9 to L-33; F-9 to D-32; F-9 to K-31; F-9 to S-30; F-9 to A-29; F-9 to Y-28; F-9 to A-27; F-9 to T-26; F-9 to A-25; F-9 to E-24; F-9 to G-23; F-9 to A-22; F-9 to D-21; F-9 to P-20; F-9 to E-19; F-9 to A-18; F-9 to D-17; F-9 to S-16; F-9 to L-15; Particular preference is given to polypeptide fragments comprising amino acid residues F-9 to R-203 of SEQ ID NO: 2, as well as to polynucleotides encoding such polypeptides. F-9 to R-203 of this SEQ ID NO: 2 polypeptide are preferably linked to S-205 to S-396 of the SEQ ID NO: 2 polypeptide. Linking can be through linkage via a linker (eg, serine, glycine, proline linkage) or via disulfide, covalent or non-covalent interactions with the antibody.

Many polynucleotide sequences, such as the EST sequence, are commercially available and accessible through the sequence database. Some of these sequences are related to SEQ ID NO: 1 and may have been commercially available prior to the inventive concept. Preferably such related polynucleotides are specifically excluded from the scope of the present invention. Enumerating all relevant sequences can be cumbersome. Thus, preferably excluded from the invention is the general formula ab, wherein a is any integer between 1 and 1660 of SEQ ID NO: 1, b is any integer between 15 and 1674, and both a and b are set forth in SEQ ID NO: 1 At least one polynucleotide comprising a nucleotide sequence corresponding to the position of the nucleotide residues, b being an integer of a + 14 or greater).

Thus, in one aspect, N-terminal deletion variants are provided by the present invention. Such variants include deletions of the first 24 or more N-terminal amino acid residues (ie, at least Met (1)-Glu (24)) to the first 115 or less N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18). Excluded are those that include the amino acid sequence shown in FIG. 1 (SEQ ID NO: 18). Alternatively, deletion of the first 24 or more N-terminal amino acid residues (ie, at least Met (1)-Glu (24)) to the first 103 or less N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18), and the like. There is an amino acid sequence shown in Figure 1 (SEQ ID NO: 18).

In another aspect, C-terminal deletion variants are provided by the present invention. Such mutants include at least the last C-terminal amino acid residue (Ser (419)) to the deletion of the last 220 or less C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18) (ie, the amino acid Val of FIG. 1 (SEQ ID NO: 18)). (199) -Ser (419) deletions), including the amino acid sequence shown in Figure 1 (SEQ ID NO: 18). The deletion will also include at least the last C-terminal amino acid residues up to the last 216 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18). The deletion will also include at least the last C-terminal amino acid residues up to the last 204 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18). The deletion will also include at least the last C-terminal amino acid residues up to the last 192 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18). The deletion will also include at least the last C-terminal amino acid residues up to the last 156 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18). The deletion will also include at least the last C-terminal amino acid residues up to the last 108 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18). The deletion will also include at least the last C-terminal amino acid residues up to the last 52 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 18).

In another aspect, deletion mutants having amino acids deleted from both N-terminal and C-terminal residues are included in the present invention. Such mutants include all combinations of the above-mentioned N-terminal deletion mutants and C-terminal deletion mutants.

The term “gene” refers to a segment of DNA involved in producing a polypeptide chain; It includes insertion sequences (introns) between individual coding segments (exons) as well as the front and rear regions (leaders and trailers) of the coding region.

The invention further relates to fragments of the isolated nucleic acid molecules described herein. A fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA, or the nucleotide sequence set forth in SEQ ID NO: 1 or 3, is about 15 nt or more in length, more preferably about useful, as a diagnostic probe and primer as discussed herein A fragment that is at least 20 nt, even more preferably at least about 30 nt, even more preferably at least about 40 nt. Of course, lengths of 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, Fragments larger than 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, or 1674nt are the nucleotide sequences of the cDNA in which these fragments are deposited or As long as they correspond to most but not all of the nucleotide sequences set forth in SEQ ID NO: 1 or 3, they are also useful in accordance with the present invention. A fragment of 20 nt or more in length means a fragment comprising at least 20 contiguous bases from the nucleotide sequence of the deposited cDNA, or from the nucleotide sequence as set forth in SEQ ID NO: 1 or 3.

Moreover, representative examples of VEGF-2 polynucleotide fragments are, for example, about nucleotides 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351- 1 or deposited clones from 400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950 or 951 There is a fragment having a sequence up to the end of the cDNA contained in. In this context, "about" includes specifically recited ranges, either large or small, on the one or both ends of the order of a few (5, 4, 3, 2 or 1) nucleotides. Preferably, these fragments encode polypeptides with biological activity.

Fragments of full length genes of the invention can be used as hybridization probes for cDNA libraries to isolate full length cDNAs and to isolate other cDNAs with high sequence similarity or similar biological activity to the genes. . Probes of this type preferably have at least about 30 bases and may contain, for example, at least 50 bases. This probe can also be used to identify cDNA clones corresponding to full length transcripts and genomic clone (s) containing the complete genes, including regulatory and promoter regions, exons and introns. One example of the screen involves separating coding regions of genes by using DNA sequences known to synthesize oligonucleotide probes. Labeled oligonucleotides having sequences complementary to the sequences of genes of the invention can be used to screen human cDNA, genomic DNA or mRNA to determine the members of the library that hybridize with the probe.

VEGF-2 “polynucleotides” can also hybridize with the sequences contained in SEQ ID NO: 1 under stringent hybridization conditions, or with the sequences of cDNA clones contained, eg, in ATCC deposits 97149 or 75698. Polynucleotides are included. "Strict hybridization conditions" means 50% formamide, 5x SSC (750mM NaCl, 75mM sodium citrate), 50mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and denatured and sheared Salmonella Incubating overnight at 42 ° C. in a solution containing 20 μg / ml of semen DNA, followed by washing the filter at 0.1 × SSC at about 65 ° C.

Also contemplated are nucleic acid molecules that hybridize with VEGF-2 polynucleotides at lower stringent hybridization conditions. The stringency of hybridization and the change in signal detection are mainly due to the formamide concentration (the lower the formamide ratio, the lower the stringency); Through manipulation of salt conditions or temperatures. For example, lower stringency conditions include 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaHPO; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% Formamide, 100 μg / ml Salmon Sperm Blocking DNA Incubating at 37 ° C. in a solution, followed by washing with 1 × SSPE, 0.1% SDS at 50 ° C. In addition, in order to achieve even lower stringency, the washing step carried out after stringent hybridization can be carried out at higher salt concentrations (eg 5X SSC).

Note that the above conditions can be changed by incorporating and / or replacing alternative blocking reagents that have been used to suppress the background in the hybridization experiment. Typical blocking reagents include the Denhardt reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available patented formulations. Incorporation of certain blocking reagents may require a change in the above-mentioned hybridization conditions due to compatibility issues therewith.

Of course, a polynucleotide that hybridizes only to a poly A + sequence (e.g., all 3 'terminal poly A + tracks of cDNAs set forth in the sequence listing) or only to complementary extensions of T (or U) residues It will not be included in the definition of “polynucleotide”, which may also hybridize with any nucleic acid molecule (eg virtually all double-stranded cDNA clones) containing the poly (A) extension or its complement. Because.

A polynucleotide that hybridizes to a "part" of a polynucleotide is at least about 15 nucleotides (nt), preferably at least about 20 nt, more preferably at least about 30 nt, most preferably about 30 to 70 nt of the reference polynucleotide. Polynucleotide (DNA or RNA) to be hybridized with. These are useful as diagnostic probes and primers as discussed above and described in detail below.

A constant “part” of a polynucleotide of “20 nt or more in length” means, for example, 20 or more contiguous from the nucleotide sequence of a reference polynucleotide (eg, deposited cDNA or nucleotide sequence as set forth in SEQ ID NO: 1). Nucleotides. Of course, hybridization only with a poly A sequence (e.g., the 3N terminal poly (A) track of VEGF-2 cDNA set forth in SEQ ID NO: 1 or SEQ ID NO: 3) or only with complementary extensions of T (or U) residues The resulting polynucleotide will not be included in the polynucleotide of the invention used to hybridize with a portion of the nucleic acid of the invention, which is any nucleic acid molecule containing a poly (A) extension or complement thereof ( For example, it can also hybridize with virtually all double-stranded cDNA clones).

The present application, whether or not encoding a polypeptide having VEGF-2 activity, comprises 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence set forth in SEQ ID NO: 1 or 3 or the nucleic acid sequence of the deposited cDNA. The present invention relates to a nucleic acid molecule exhibiting the same rate. This is true to those skilled in the art, even if a particular nucleic acid molecule does not even encode a polypeptide with VEGF-2 activity, for example as a hybridization probe or a polymerase chain reaction (PCR) primer. This is because the method is still known. Uses of the nucleic acid molecules of the invention that do not encode polypeptides having VEGF-2 activity include, inter alia, (1) isolation of the VEGF-2 gene or allelic variants thereof in the cDNA library; (2) Mid-term chromosomal expansion to provide accurate chromosomal location of the VEGF-2 gene as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988). In situ hybridization with water (eg, "FISH"); There is a northern blot analysis to detect VEGF-2 mRNA expression in specific tissues.

In practice, however, it exhibits at least 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence set forth in SEQ ID NO: 1 or 3 or the nucleic acid sequence of deposited cDNA encoding a polypeptide having VEGF-2 protein activity. Nucleic acid molecules are preferred. "Polypeptide with VEGF-2 activity" means a polypeptide that exhibits VEGF-2 activity in a particular biological assay. For example, VEGF-2 protein synthesis can be measured using, for example, mitotic assays and endothelial cell migration assays. Olofsson et al., Proc. Natl. Acad. Sci. USA 93: 2576-2581 (1996) and Joukov et al., EMBO J. 5: 290-298 (1996).

Of course, due to genetic code denaturation, one of ordinary skill in the art would be 90%, 95%, 96%, 97%, 98% or 99% of the deposited cDNA or the nucleic acid sequence set forth in SEQ ID NO: 1 or 3. It will be immediately appreciated that multiple nucleic acid molecules having sequences exhibiting the same identity rates will encode polypeptides having "VEGF-2 protein activity". In fact, since all of the variant variants of these nucleotide sequences encode the same polypeptide, it would be apparent to one skilled in the art even if this was not done with the comparative assays mentioned above. For such nucleic acid molecules that are not denaturing variants, it will be further appreciated in the art that a significant number will also encode polypeptides having VEGF-2 protein activity. This is because those skilled in the art are fully aware of amino acid substitutions (eg, replacing one aliphatic amino acid with a second aliphatic amino acid) that perform substantially less or less protein function.

For example, instructions on how to make morphologically asymptomatic amino acid substitutions are described in Bowie, J.U. et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 247: 1306-1310 (1990), in which the author surprisingly accepts amino acid substitutions. Instructed.

Accordingly, the present invention provides polynucleotides encoding the polypeptides set forth in SEQ ID NO: 2 or 4 as well as fragments thereof having at least 30 bases, preferably at least 50 bases, and at least 70%, preferably at least 90%, more preferably Is a polynucleotide exhibiting an identical rate of at least 95%, 96%, 97%, 98% or 99%; And polypeptides encoded by such polynucleotides.

"Equal" itself has a recognized meaning in the art and can be calculated using published techniques. COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed. Oxford University Press, New York, (1988); BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Giffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, (1991). Although there are numerous ways to measure the same rate between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art. See Carillo, H., and Lipton, D. SIAM J. Applied. Math. 48: 1073 (1988). Methods commonly used to determine the identity or similarity between two sequences include those described in "Guide to Huge Computers", Martin J. Bishop, ed., Academic Press, San Diego, (1994), and Carillo, H., and Lipton, D., SIAM J. Applied Math. 48: 1073 (1988), but is not limited to such. Methods for aligning polynucleotides or polypeptides can be found in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF, et al. , J. Molec. Biol. 215: 403 (1990)), best-fit program [Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711 (Smith and Using the local homology algorithm of Waterman, Advances in Applied Mathematics 2: 482-489 (1981)). A polynucleotide having a reference polynucleotide sequence of the present invention and a nucleotide sequence representing, for example, at least 95% "identity" means that each polynucleotide sequence of each 100 nucleotides of the reference nucleotide sequence that encodes a VEGF-2 polypeptide It means that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that it may contain up to 5 point mutations. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, only 5% or less of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or the total nucleotides in the reference sequence Nucleotide numbers up to 5% of can be inserted into the reference sequence. The interrogative sequence can be the complete sequence shown in SEQ ID NO: 1, an ORF (open reading frame), or any fragment specified as described herein.

In practice, it is convenient to use a known computer program to determine whether a particular nucleic acid molecule or polypeptide exhibits at least 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence of the present invention. You can decide. A preferred method for determining the overall best match between a questionable sequence (a sequence of the invention) and a subject sequence, also referred to as overall sequence alignment, is determined using a FASTDB computer program based on an algorithm of Brutlag et al. Brutlag et al. (1990) Comp. App. Biosci. 6: 237-245. In sequence alignment, both the questionable and subject sequences are DNA sequences. RNA sequences can be compared by first converting U to T. This overall sequence alignment result is percent identity. Preferred parameters used for FASTDB alignment of DNA sequences to calculate percent identity are as follows: matrix = unitary, k-tuple = 4, mismatch penalty = 1, joining penalty = 30, random group length = Length of subject nucleotide sequence whether 0, cutoff score = 1, gap penalty = 5, gap size penalty = 0.05, window size = 500 or shorter.

If the subject sequence is shorter than the questionable sequence due to 5 'or 3' deletions, not due to internal deletions, the results must be corrected manually. This is because the FASTDB program does not consider 5 'and 3' cleavage of the subject sequence when estimating the identity rate. For a subject sequence whose 5 'or 3' end is cut relative to the interrogative sequence, the base number of the interrogative sequences 5 'and 3' of the subject sequence, which are not matched and not aligned, is calculated as the total base% of the interrogative sequence. Correct the same rate. Whether a particular nucleotide is matched / aligned is determined by the result of the FASTDB sequence alignment. This ratio is then subtracted from the percent percent calculated by the FASTDB program using the parameters specified above to reach a final percent percent score. This corrected score is used for the purposes of the present invention. Only bases outside the 5 'and 3' bases of the subject sequence as indicated by the FASTDB alignment, which are not matched / aligned with the questionable sequence, are calculated to manually adjust the percent identity score.

For example, a 90 base subject sequence is aligned with a 100 base question sequence to determine percent identity. As deletions occur at the 5 'end of the subject sequence, the FASTDB alignment does not show a match / alignment of the first 10 bases at the 5' end. 10 unpaired bases represent 10% of the sequence (the number of bases at the 5 'and 3' ends does not match the total number of bases in the questionable sequence) and 10% was calculated by the FASTDB program. It is reduced from the same percentage (%) score. If the remaining 90 bases were a perfect match, the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base question sequence. At this time, the deletion is an internal deletion such that there is no base on the 5 'or 3' of the sequence of interest that is not matched / aligned with the questionable sequence. In this case, the percent equality calculated by FASTDB is not manually corrected. Once again, only the 5 'and 3' bases of the subject sequence that do not match / align with the questionable sequence are manually corrected. No other manual calibration is made for the purposes of the present invention.

A polypeptide having an amino acid sequence exhibiting an "equal rate" of, for example, 95% or more of the questionable amino acid sequence of the present invention means that the amino acid sequence of such a polypeptide of interest is 5 or less for each 100 amino acids of the questionable amino acid sequence. It is meant the same as the questionable sequence except that it may include amino acid modifications. In other words, to obtain a polypeptide having an amino acid sequence that exhibits at least 95% identity with a questionable amino acid sequence, up to 5% of the amino acid residues in the sequence of interest can be inserted, deleted, or substituted with another amino acid. Such modification of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between these terminal positions, interspersed among the residues in the reference sequence or in one or more consecutive groups within the reference sequence. Interspersed.

In practical cases, specific polypeptides may comprise 90%, 95%, 96%, 97%, nucleotide sequences of the invention, for example the amino acid sequences set forth in Table 1, or amino acid sequences encoded by deposited cDNA clones, Whether or not the same rate is 98% or 99% or more can be conveniently determined using a known computer program. Preferred methods for determining the overall best match between a questionable sequence (a sequence of the invention) and a subject sequence, sometimes referred to as overall sequence alignment, can be determined using a FASTDB computer program based on the algorithm of Brutlag et al. See Brutlag et al. (1990) Comp. App. Biosci. 6: 237-245. In sequence alignment, the interrogative and subject sequences are both nucleotide sequences or both amino acid sequences. This overall sequence alignment result is percent identity. Preferred parameters used for FASTDB amino acid alignment are: matrix = PAM 0, k-tuple = 2, mismatch penalty = 1, joining penalty = 20, random group length = 0, cutoff score = 1, window size = Length of the subject amino acid sequence, whether sequence length, gap penalty = 5, gap size penalty = 0.05, window size = 500 or shorter.

If the sequence of interest is shorter than the questionable sequence due to N- or C-terminal deletions, not due to internal deletions, the results must be corrected manually. This is because the FASTDB program does not take into account the N- and C-terminal truncations of the subject sequence when calculating the overall identity rate. For a subject sequence whose N- or C-terminus is truncated relative to the interrogative sequence, the number of residues of the intervening sequence that is the N- and C-terminus of the subject sequence that do not match and are not aligned with the corresponding subject residue is determined by The same rate is corrected by calculating as total base%. Whether a particular residue is matched / aligned is determined by the result of the FASTDB sequence alignment. This ratio is then subtracted from the percent percent calculated by the FASTDB program using the parameters specified above to reach a final percent percent score. This final equality score is used for the purposes of the present invention. Only residues on the N- and C-terminus of the subject sequence that are not matched / aligned with the questionable sequence are considered to manually adjust the percent identity score. That is, only questionable residues at positions beyond the furthest N- and C-terminal residues of the subject sequence are considered.

For example, a 90% amino acid residue subject sequence is aligned with a 100 residue question sequence to determine percent identity. As deletions occur at the N-terminus of the subject sequence, the FASTDB alignment does not indicate a match / alignment of the first 10 residues at the N-terminus. 10 unpaired residues represent 10% of the sequence (the number of residues at the N- and C-terminals does not match the total number of residues in the questionable sequence) and 10% was calculated by the FASTDB program. It is reduced from the same percentage (%) score. If the remaining 90 residues were a perfect match, the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue questionable sequence. At this time, the deletion is an internal deletion such that there are no residues on the N- or C-terminus of the sequence of interest that are not matched / aligned with the sequence in question. In this case, the percent equality calculated by FASTDB is not manually corrected. Once again, as shown in the FASTDB alignment, only residues at positions outside the N- and C-terminus of the subject sequence that do not match / align with the questionable sequence are manually corrected. No other manual calibration is made for the purposes of the present invention.

VEGF-2 polypeptide

The invention further relates to polypeptides having the inferred amino acid sequence of FIG. 1 or 2, or amino acid sequence encoded by deposited cDNA, and fragments, homologs and derivatives of such polypeptides.

The terms “fragment”, “derivative” and “cognate” when referring to the polypeptide of FIG. 1 or 2 or the polypeptide encoded by the deposited cDNA retain the conserved motif of the VEGF protein as shown in FIG. 3. It means a polypeptide having essentially the same biological function or activity.

In the present invention, "polypeptide fragment" refers to a short amino acid sequence encoded by cDNA contained in the clone contained in or deposited in SEQ ID NO: 2. Protein fragments may be "free-standing", or fragments thereof may be included in larger polypeptides that form one part or region most preferably as a single contiguous region. Representative examples of polypeptide fragments of the invention include, for example, amino acid numbers 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161- 180, 181-200, 201-220, 221-240, 241-260, 261-280, or 281 to the end of the coding region. Moreover, polypeptide fragments may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 or more amino acids in length. In this regard, “about” includes particularly mentioned ranges at which either amino acid at either or both ends is greater or smaller, such as about 5, 4, 3, 2 or 1 amino acids.

Preferred polypeptide fragments include the secreted VEGF-2 protein as well as the mature form. Further preferred polypeptide fragments include mature forms having a series of residues deleted from the secreted VEGF-2 protein or amino or carboxy termini, or both. For example, any number of amino acids ranging from 1 to 60 can be deleted from the secreted VEGF-2 polypeptide or the amino terminus of the mature form. Similarly, any number of amino acids in the range of 1 to 30 can be deleted from the secreted VEGF-2 polypeptide or mature form of the carboxy terminus. Moreover, any combination of the above amino and carboxy terminal deletions is preferred. Similarly, polynucleotide fragments encoding these VEGF-2 polypeptides are also preferred.

In addition, alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet forming regions, turn and turn-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions Preference is given to VEGF-2 polypeptides and polynucleotides characterized by structural or functional domains, such as fragments comprising regions, flexible regions, surface forming regions, substrate binding regions and high antigenic index regions. Polypeptide fragments of SEQ ID NO: 2 that fall within conserved domains are particularly contemplated in the present invention (see Figure 2). Moreover, polynucleotide fragments encoding these domains are contemplated.

Other preferred fragments are biologically active VEGF-2 fragments. A biologically active fragment is one that is not necessarily identical to the activity of the VEGF-2 polypeptide but exhibits similar activity. The biological activity of such fragments may include enhancing desired activity or undesirably lowering activity.

The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides, preferably recombinant polypeptides.

It will be appreciated in the art that several amino acid sequences of the VEGF-2 polypeptide may vary without materially affecting the structure or function of the protein. If such sequence differences are considered, it should be remembered that there is an important part on the protein that determines activity.

Accordingly, the present invention further encompasses variants of VEGF-2 polypeptides that exhibit substantial VEGF-2 polypeptide activity or comprise regions of the VEGF-2 protein, such as the protein portions discussed below. Such mutants include deletions, inserts, conversions, repeats and type substitutions. As indicated above, guidance regarding amino acid changes that are morphologically asymptomatic is described in Bowie, J.U. et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 247: 1306-1310 (1990).

Thus, fragments, derivatives or homologs of the polypeptide of FIG. 1 or 2, or a polypeptide encoded by a deposited cDNA, may be: (i) an amino acid residue (preferably with or without conserving one or more amino acid residues) Is a conserved amino acid residue), which may or may not be encoded by a genetic code; (Ii) at least one amino acid residue comprises a substituent group; (Iii) the mature polypeptide is fused with another compound, such as a compound (eg, polyethylene glycol) to increase the half-life of the polypeptide; (Iii) a sequence or leader or secretory sequence, or proprotein sequence, wherein the additional amino acid is used for purification of a mature polypeptide, eg, a mature polypeptide; Or (v) contains fewer than the amino acid residues set forth in SEQ ID NO: 2 or 4, possesses a conserved motif and still retains the active characteristics of the VEGF family of polypeptides. Such fragments, derivatives and homologs are considered to be within the scope of the art from the teachings herein.

Of particular interest is the substitution of charged amino acids with other charged amino acids and with neutral or negatively charged amino acids. Due to the latter, proteins with reduced positive charges are produced to improve the characteristics of the VEGF-2 protein. It is highly desirable to inhibit aggregation. Aggregation of proteins not only results in loss of activity, but also causes problems when preparing pharmaceutical preparations, because the aggregates may be immunogenic (Pinckard, et al., Clin. Exp. Immunol. 2: 331-340 (1967); Robbins, et al., Diabetes 36: 838-845 (1987); Cleland, et al., Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993).

Substitution of amino acids may also alter binding selectivity to cell surface receptors. Ostade et al., Nature 361: 266-268 (1993) describe specific mutations that selectively bind TNF-a to only one of two known types of TNF receptors. Thus, VEGF-2 of the present invention may comprise one or more amino acid substitutions, deletions or adducts from natural mutations or human manipulations.

As indicated, minimal changes are preferred, such as conservative amino acid substitutions that do not substantially affect the folding or activity of the protein (see Table 1).

Conservative Amino Acid Substitutions Aromatic Phenylalanine tryptophan tyrosine Hydrophobic Leucine isoleucine valine polarity Glutamine asparagine Basic Arginine lysine histidine acid Aspartic Acid Glutamic Acid etc Alanine Serine Threonine Methionine Glycine

Of course, those skilled in the art will appreciate that the number of amino acid substitutions may depend on a number of factors, including those mentioned above. Generally speaking, the number of substitutions for a given VEGF-2 polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3 or more.

Amino acids in the VEGF-2 protein of the invention essential for function are known in the art, such as site directed mutations or alanine-scanning mutations. Cunningham and Wells, Science 244: 1081-1085 (1989) ] Can be identified. The latter method introduces a single alanine mutation at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vivo or in vitro proliferative activity. Sites important for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling. Smith et al., J. Mol. Biol. 224: 899-904 (1992) and de Vos et al. Science 255: 306-312 (1992).

Polypeptides and polynucleotides of the invention are preferably provided in isolated form, and are preferably homogeneously purified.

The term "isolated" refers to the removal of the substance from its original environment (eg, the natural environment if present naturally). For example, natural polynucleotides or polypeptides present in living animals are not isolated, but identical polynucleotides or polypeptides isolated from some or all of the materials present together in a natural system are isolated. Such polynucleotides may be part of a vector and / or such polynucleotides or polypeptides may be part of a composition and are still isolated in that such vector or composition is not part of its natural environment.

In certain embodiments, polynucleotides of the invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb or 7.5 kb in length. In further embodiments, the polynucleotides of the present invention comprise at least 15 contiguous nucleotides of the VEGF-2 coding sequence, but do not include some or all of the VEGF-2 introns. In another embodiment, the nucleic acid comprising the VEGF-2 coding sequence does not contain the coding sequence of the genomic flanking gene (ie, 5 'or 3' for the VEGF-2 gene in the genome).

Polypeptides of the present invention include polypeptides of SEQ ID NOs: 2 and 4 (particularly mature polypeptides) as well as polypeptides of at least 70% (preferably at least 70% identity), and more preferably polypeptides of SEQ ID NOs: 2 and 4 And polypeptides having at least 90% similarity (more preferably at least 95% identity), even more preferably at least 95% similarity (more preferably at least 90% identity) with the polypeptides of SEQ ID NOS: 2 and 4 And also generally include portions of a polypeptide having said portion of a polypeptide containing at least 30 amino acids, more preferably at least 50 amino acids.

As is known in the art, the “similarity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutions with the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention can be used to prepare corresponding full length polypeptides by peptide synthesis, so the fragments can be used as intermediates to produce full length polypeptides. Fragments or portions of the polynucleotides of the invention can be used to synthesize full length polynucleotides of the invention.

Polypeptides of the invention include polypeptides encoded by deposited cDNA including a leader; Mature polypeptide (ie, mature protein) encoded by deposited cDNA except the leader; A polypeptide comprising an amino acid of about -23 to about 396 in SEQ ID NO: 2; A polypeptide comprising an amino acid of about -22 to about 396 in SEQ ID NO: 2; A polypeptide comprising about 1 to about 396 amino acids in SEQ ID NO: 2; And polypeptides exhibiting an identical rate of at least 95%, more preferably at least 96%, 97%, 98% or 99% of the polypeptides mentioned above and also containing at least 30 amino acids, more preferably at least 50 amino acids. Part of the polypeptide is included.

Fusion protein

Any VEGF-2 polypeptide can be used to generate the fusion protein. For example, when a VEGF-2 polypeptide is fused with a second protein, it can be used as an antigenic tag. Antibodies generated against VEGF-2 polypeptides can be used to indirectly direct a second protein by binding to VEGF-2. Moreover, because secreted proteins can target intracellular locations based on signal trafficking, VEGF-2 polypeptides can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused with VEGF-2 polypeptides include heterologous signal sequences as well as other heterologous functional regions. Such fusion is not necessary to indicate, but can occur through linker sequences.

Moreover, the fusion protein can be engineered to improve the characteristics of the VEGF-2 polypeptide. For example, certain regions of additional amino acids, especially charged amino acids, may be added to the N-terminus of the VEGF-2 polypeptide to enhance stability and persistence in the host cell during purification or during subsequent manipulation and storage. In addition, peptide residues may be added to the VEGF-2 polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the VEGF-2 polypeptide. Adding peptide residues to facilitate manipulation of polypeptides is a common technique well known in the art.

Moreover, VEGF-2 polypeptides, including fragments, and specifically epitopes, may be combined with portions of the constant region of immunoglobulins (IgG) to produce chimeric polypeptides. These fusion proteins promote purification and increase half-life in vivo. The reported example describes chimeric proteins consisting of the first two regions of human CD4-polypeptide and the various regions of the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP A 394,827; Traunecker et al., Nature 331: 84-86 (1988). Fusion proteins with disulfide-linked chimeric structures (due to IgG) may also be more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone. Fountoulakis et al., J. Biochem. 270: 3958-3964 (1995).

Similarly, EP-A-O 464 533 (Canada Corresponding Publication No. 2045869) describes fusion proteins comprising various portions of the constant region of an immunoglobulin molecule with another human protein or part thereof. In many cases, the Fc portion of the fusion protein is quite advantageous for use in therapy and diagnostics, for example, so that the pharmacodynamic properties are improved (EP-A 0232 262). On the other hand, after expressing, detecting and purifying the fusion protein, it would be desirable to remove the Fc moiety. For example, when the fusion protein is used as an antigen for immunization, the Fc moiety can interfere with treatment and diagnosis. In drug development, for example, human proteins such as hIL-5 were fused with the Fc moiety for high-output screening assays to identify antagonists of hIL-5. See, eg, D. Bennett et al., J. Molecular. Recognition 8: 52-58 (1995) and K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995).

Furthermore, the VEGF-2 polypeptide can be fused with a peptide that facilitates the purification of a marker sequence, eg, VEGF-2. In a preferred embodiment, the marker amino acid sequence is a tag provided in a hexa-histidine peptide, such as a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311) among many commercially available products. See Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for example, hexa-histidine provides convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to epitopes derived from influenza hemagglutinin proteins described in Wilson et al., Cell 37: 767 (1984).

Thus, any of these fusions can be engineered using VEGF-2 polynucleotides or polypeptides.

Biological Activity of VEGF-2

VEGF-2 polynucleotides and polypeptides can be used in assays to test one or more biological activities. Where VEGF-2 polynucleotides and polypeptides are active in certain assays, it is believed that VEGF-2 may be involved in diseases associated with biological activity. Thus, VEGF-2 can be used to treat such associated diseases.

Immune activity

VEGF-2 polypeptides or polynucleotides may be useful for treating deficiencies or disorders of the immune system by activating or inhibiting the proliferation, differentiation or recruitment of immune cells (chemotaxis). Immune cells grow through a process called hematopoiesis, producing myeloid cells (platelets, red blood cells, neutrophils and macrophages) and lymphoid cells (B and T lymphocytes) from pluripotent liver cells. The etiology of these immune deficiencies or disorders can be genetic, somatic, for example acquired (eg, caused by chemotherapy or toxin) autoimmune disorder or cancer, or infectious. Moreover, VEGF-2 polynucleotides or polypeptides can be used as markers or detection agents for certain immune system diseases or disorders.

VEGF-2 polynucleotides or polypeptides may be useful for treating or detecting deficiencies or disorders of hematopoietic cells. VEGF-2 polynucleotides or polypeptides can be used to increase the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, to treat disorders associated with the reduction of certain (or many) types of hematopoietic cells. Examples of immunological deficiencies include blood protein disorders (e.g. agammagglobulinemia or selective immunoglobulin deficiency), ataxia capillary dilatation, various common immunodeficiencies, Digeorge syndrome, HIV infection, HTLV-BLV infection, lymphocytes Adhesion bonds, lymphocytosis, phagocytic bactericidal insufficiency, severe complications of immunodeficiency (SCIDs), Wiskott-Aldrich disorders, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, VEGF-2 polynucleotides or polypeptides can be used to modulate hemostasis (stop bleeding) activity or thrombus collapse activity (coagulation formation). By enhancing hemostatic or thrombolytic activity, VEGF-2 polynucleotides or polypeptides can cause blood clotting disorders (e.g. afibrinogenemia, factor deficiency), platelet disorders (e.g. thrombocytopenia) or traumas resulting from injury, surgery or other causes It can be used to treat Alternatively, VEGF-2 polynucleotides or polypeptides capable of lowering hemostatic or thrombolytic activity can be used to inhibit or dissolve blood clots, which are important for the treatment of heart attack (infarction), paralysis or scarring.

VEGF-2 polynucleotides or polypeptides may be useful for treating or detecting autoimmune disorders. Many autoimmune disorders result from the improper recognition of their own material as foreign by immune cells. This inadequate recognition results in an immune response that results in the destruction of host tissue. Thus, administration of VEGF-2 polynucleotides or polypeptides that can inhibit the immune response, particularly the proliferation, differentiation or chemotaxis of T cells, may be an effective treatment for preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated or detected by VEGF-2 include, but are not limited to: Addison disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergy Sexual encephalomyelitis, glomerulonephritis, goodpasture syndrome, Graves' disease, multiple sclerosis, myasthenia gravis, neuritis, conjunctivitis, bullous swelling, blistering, multiple endocrine disease, purpura, Reuters disease, Stiff-man Man) disease, autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary artery inflammation, Guillain-Barre syndrome, insulin dependent diabetes and autoimmune inflammatory eye disease.

Similarly, allergic reactions and diseases such as asthma (particularly allergic asthma) or other respiratory disorders can be treated with VEGF-2 polynucleotides or polypeptides. Moreover, VEGF-2 can be used to treat anaphylaxis, hypersensitivity to antigenic molecules or blood group incompatibility.

VEGF-2 polynucleotides or polypeptides can also be used to treat and / or prevent organ rejection or graft-versus-host disease (GVHD). Organ rejection is caused by the destruction of host immune cells of the transplanted tissue via an immune response. Similarly, the immune response is also involved in GVHD, but in this case, the transplanted foreign immune cells destroy the host tissue. Administration of VEGF-2 polynucleotides or polypeptides that inhibit the immune response, particularly the proliferation, differentiation or chemotaxis of T cells, may be an effective treatment for preventing organ transplantation or GVHD. Similarly, VEGF-2 polynucleotides or polypeptides may be used to modulate inflammation. For example, VEGF-2 polynucleotides or polypeptides can inhibit the proliferation and differentiation of cells involved in the inflammatory response. These molecules include inflammation associated with infection (eg, septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemic-perfusion injury, endotoxin lethality, arthritis, complement-mediated hypersensitivity rejection, nephritis, cytokine or chemokine-induced It can be used to treat chronic and acute conditions of inflammatory diseases, including lung damage, inflammatory bowel disease, Crohn's disease, or diseases resulting from overproduction of cytokines such as TNF or IL-1.

Hyperproliferative disorder

VEGF-2 polynucleotides or polypeptides can be used to treat or detect hyperproliferative disorders, including neoplasms. VEGF-2 antagonist polynucleotides or polypeptides can inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, the VEGF-2 antagonist polynucleotide or polypeptide can proliferate other cells that can inhibit hyperproliferative disorders.

For example, hyperproliferative disorders can be treated by increasing the immune response, in particular by increasing the antigenic nature of the hyperproliferative disorder, or by proliferating, differentiating or mobilizing T cells. This immune response can be increased by changing the existing immune response or initiating a new immune response. Alternatively, lowering the immune response may also be a method of treating a hyperproliferative disorder, such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detected by VEGF-2 antagonist polynucleotides or polypeptides include, but are not limited to, neoplasms localized to the following: abdomen, bone, breast, digestive system, liver, pancreas. , Peritoneum, endocrine gland (adrenal, epithelial, pituitary, testes, ovary, thymus, thyroid), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, rib cage and Genitourinary system.

Similarly, other hyperproliferative disorders may be treated or detected by VEGF-2 antagonist polynucleotides or polypeptides. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sezary syndrome, Waldenstron macroglobulinemia, Gosch disease, histiocytosis, and other hyperproliferative diseases except neoplasia localized in the organ systems listed above.

Infectious disease

VEGF-2 polynucleotides or polypeptides can be used to treat or detect infectious diseases. For example, infectious diseases can be treated by increasing the immune response, in particular by increasing the proliferation and differentiation of B and / or T cells. This immune response can be increased by changing the existing immune response or initiating a new immune response. Alternatively, the VEGF-2 polynucleotide or polypeptide can also directly inhibit an infectious disease without necessarily inducing an immune response.

One example of an infectious agent that can cause a disease or syndrome that can be treated or detected by a VEGF-2 polynucleotide or polypeptide is a virus. Examples of viruses include, but are not limited to, the following DNA and RNA viral classes: arboviruses, adenoviruses, arenaviruses, arteriviruses, birnaviridae, buniaviridae, calciviridae, sir Corbyridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (e.g. Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g. Paramyxoviridae , Morbivirus, Lavdoviridae), orthomyxoviridae (e.g. influenza), Paphobaviridae, Parvoviridae, Picornaviridae, Foxviridae (e.g. Small Fox or Vaccinia), Leo Sundae (eg rotavirus), Retroviridae (HTLV-1, HTLV-II, lentivirus) and Togaviridae (eg rubyvirus). Viruses within these classes can cause a variety of diseases or symptoms, including but not limited to: arthritis, bronchitis, encephalitis, eye inflammation (eg, conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A , B, C, E, chronic active, delta), meningitis, opportunistic infections (e.g. AIDS), pneumonia, Burkitt's lymphoma, chickenpox, hemorrhagic fever, margin, mumps, parainfluenza, rabies, cold, polio, leukemia , Rubella, venereal disease, dermatosis (eg, kaposi, pancreas) and viremia. VEGF-2 polynucleotides or polypeptides can be used to treat or detect any of these syndromes or diseases.

Similarly, bacterial or fungal agents that can cause diseases or syndromes that can be treated or detected by VEGF-2 polynucleotides or polypeptides include the following Gram-negative and Gram-positive bacterial families and fungi, It is not limited to: actinomycetales (e.g. Corynebacterium, Mycobacterium, Norcardia), aspergillosis, basilisae (e.g. Anthrax, Clostridium), bacteriodase, blas Tomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Cosidioomycosis, Cryptococosis, Dermatophycose, Enterobacteriaceae (Clebciella, Salmonella, Cera Tia, Yersinia), Erysipelotrix, Helicobacter, Leginelosis, Leptospirosis, Listeria, Mycoplasma Matales, Neisseria ceae (e.g. Acinetobacter, Gonorrhea, Menigococal), Pasteurella bird infections (eg Actinobacillus, Hemophilus, Pasteurella), Pseudomonas, Rickettsiaea, Chlamydiaceae, Ciphylis and Staphylococcal. These bacterial and fungal families can cause diseases or syndromes including but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (eg AIDS related infections) ), Prostatitis, prosthetic-related infections, Reuters disease, respiratory infections, for example pertussis or sinusitis, sepsis, limea disease, cat-scratch disease, dysentery, epithelial fever, food poisoning, typhoid, pneumonia, gonorrhea, meningitis, Chlamydia, syphilis, diphtheria, leprosy, tuberculosis, tuberculosis, lupus, botulism, necrosis, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted disease, skin disease (e.g., cellulitis, dermacycos), toxins, urinary tract infections, trauma infection. VEGF-2 polynucleotides or polypeptides can be used to treat or detect any of these syndromes or diseases.

Moreover, parasitic agents that can cause diseases or syndromes that can be treated or detected by VEGF-2 polynucleotides or polypeptides include, but are not limited to: amebiasis, vavesiosis, kosi Diossis, Cryptosporidiosis, Dientamobiasis, Dourine, Ectoparasitic, Giardiasis, Helmintiasis, Leishmaniasis, Teleriasis, Toxoplasmosis, Tripanosomiasis and Trichomonas. These parasites can cause a variety of diseases or syndromes, including but not limited to: amelioration, Tsugamus disease, eye infections, intestinal diseases (e.g., dysentery, lamiparasminosis), liver disease, lung disease, opportunism Infections (eg AIDS related diseases), malaria, pregnancy complications and toxoplasmosis disease. VEGF-2 polynucleotides or polypeptides can be used to treat or detect any of these syndromes or diseases.

Preferably, treatment with a VEGF-2 polynucleotide or polypeptide administers an effective amount of VEGF-2 polypeptide to a patient, removes a cell from the patient, and provides the cell with a VEGF-2 polynucleotide, Treated cells can be performed by reinjecting (ex vivo treatment) to a patient. Moreover, VEGF-2 polynucleotides or polypeptides can be used as antigens in vaccines that induce an immune response to an infectious disease.

play

VEGF-2 polynucleotides or polypeptides can be used to differentiate, proliferate, and attract cells, causing tissue regeneration (Sci. 276: 58-87 (1997)). Tissue regeneration may include congenital defects, injuries (traumas, burns, wounds or ulcers), aging, diseases (e.g., osteoporosis, osteocartritis, periodontal, liver failure), surgery (including cosmetic plastic surgery), fibrosis, reperfusion injury, or systemic It can be used to repair, replace or prevent tissue damaged by cytokine damage.

Tissues that can be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidneys, skin, endothelium), muscles (smooth muscle, skeletal muscle or myocardium), blood vessels (including vascular endothelial), lymphoid (lymphatic) Endothelial), nerve, hematopoietic and skeletal (bone, cartilage, tendon, and ligament) tissues. Preferably, regeneration occurs with or without scar formation. Regeneration may also include angiogenesis.

Moreover, VEGF-2 polynucleotides or polypeptides can increase the regeneration of tissue that is difficult to treat. For example, increased tendon / ligament regeneration will shorten recovery time after injury. The VEGF-2 polynucleotides or polypeptides of the invention may also be used prophylactically to avoid damage. Diseases that can be treated include tendonitis, humerus tunnel syndrome and other tendon or ligament defects. Further examples of tissue regeneration of untreated trauma include ulcers associated with compressive ulcers, vascular insufficiency, surgery, and injury trauma.

Neuronal and brain tissue may also be regenerated by using VEGF-2 polynucleotides or polypeptides to proliferate and differentiate neurons. Diseases that can be treated using this method include central and peripheral nervous system diseases, neuropathy, or mechanical and injury disorders such as spinal cord disorders, brain damage, cerebrovascular disease, and stroke. Specifically, using VEGF-2 polynucleotides or polypeptides, peripheral nerve damage, peripheral neuropathy (eg, resulting from chemotherapy or other medical treatments), localized neuropathy and central nervous system diseases (eg, Alzheimer's disease, Parkinson's) Disease, Huntington's disease, amyotrophic lateral sclerosis and Shy-Dragger syndrome).

Chemotaxis

VEGF-2 polynucleotides or polypeptides may have chemotactic activity. Chemotactic molecules attract cells (eg monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and / or endothelial cells) to specific sites in the body, such as inflammation, infection, or hyperproliferation sites. Or mobilize. The cells so mobilized then fight and / or heal specific damage or abnormalities.

VEGF-2 polynucleotides or polypeptides can increase chemotactic activity of certain cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or all immune system disorders by increasing the number of cells targeted to specific locations in the body. For example, chemotactic molecules can be used to treat trauma and tissue damage by attracting immune cells to the damaged site. As chemotactic molecules, VEGF-2 can also attract fibroblasts that can be used to treat trauma.

It is also contemplated that the VEGF-2 polynucleotide or polypeptide may inhibit chemotactic activity. These molecules can also be used to treat disorders. Thus, VEGF-2 polynucleotides or polypeptides can be used as inhibitors of chemotaxis.

Combined activity

VEGF-2 polypeptides can be used to screen for molecules that bind VEGF-2 or molecules for which VEGF-2 is bound. Binding of VEGF-2 and the molecule can activate (inhibitor), increase, inhibit (antagonist) or decrease the activity of VEGF-2 or the bound molecule. Examples of such molecules are antibodies, oligonucleotides, proteins (eg receptors) or small molecules.

Preferably, the molecule is closely related to a natural ligand of VEGF-2, eg, a fragment of such ligand, or a natural substrate, ligand, structural or functional mimetic. Coligan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)]. Similarly, the molecule is closely related to the native receptor to which VEGF-2 is bound (ie, Flt-4), or at least a fragment of the receptor (eg, active site) that can be bound by VEGF-2. have. In either case, the molecule can be appropriately designed using known techniques. Preferably, screening for these molecules involves generating suitable cells that express VEGF-2 as a secreted protein or on the cell membrane. Preferred cells include mammalian, yeast, drosophila or E. Cells from E. coli are included. It is then desirable to contact the cells expressing VEGF-2 (or the cell membrane containing the expressed polypeptide) with potentially a test compound containing the molecule to observe binding, stimulation or inhibition of VEGF-2 or the molecule. .

This assay can simply test bind candidate compounds to VEGF-2, where binding is detected by a label or in an assay involving competition with a labeled competitor. In addition, the assay can test whether candidate compounds produce a signal generated by binding to VEGF-2.

Alternatively, this assay can be performed using cell-free preparations, polypeptides / molecules immobilized on a solid support, chemical libraries, or natural product mixtures. This assay simply includes mixing the candidate compound with a VEGF-2 containing solution, measuring VEGF-2 / molecular activity or binding and comparing VEGF-2 / molecular activity or binding to a standard. do.

Preferably, the ELISA assay may use monoclonal or polyclonal antibodies to determine VEGF-2 levels or activity in certain samples (eg, biological samples). This antibody can measure VEGF-2 levels or activity by binding directly or indirectly to VEGF-2 or by competing with VEGF-2 for a particular substrate.

All of these assays can be used as diagnostic or prognostic markers. Molecules discovered using these assays can be used to treat diseases or to produce specific results (eg, vascular growth) in patients by activating or inhibiting VEGF-2 / molecules. Moreover, these assays enable the discovery of agents that can inhibit or enhance the production of VEGF-2 from suitably engineered cells or tissues.

Accordingly, the present invention includes a method of identifying a compound that binds to VEGF-2, comprising (a) culturing the candidate binding compound with VEGF-2 and (b) determining whether binding occurs. do. Moreover, the present invention comprises the steps of (a) culturing the candidate compound with VEGF-2; (b) assaying the biological activity; And (c) determining whether the biological activity of VEGF-2 has changed.

Other active

VEGF-2 polypeptides or polynucleotides can increase or inhibit differentiation or proliferation of embryonic liver cells beyond hematopoietic lineage as discussed above.

VEGF-2 polypeptides or polynucleotides can be used to control mammalian features such as weight, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size and appearance (eg, plastic surgery). Similarly, VEGF-2 polypeptides or polynucleotides can be used to regulate mammalian metabolism affecting catabolism, assimilation, processing, utilization and energy storage.

VEGF-2 polypeptides or polynucleotides are characterized by virorhythms, cardiac rhythms, depression (including depressive disorders), tendency to intensify, resistance to pain, regenerative capacity (preferably by activin or inhibin-like activity) ) Can be used to alter the mental or physical state of a mammal by affecting hormone or endocrine levels, appetite, libido, memory, stress or other cognition.

VEGF-2 polypeptides or polynucleotides may also be used as food additives or preservatives, for example, to increase or decrease storage capacity, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other nutritional components. have.

Vector, host cell and protein production

The invention also provides recombinant vectors comprising the isolated nucleic acid molecules of the invention and host cells containing such recombinant vectors, as well as methods of making such vectors and host cells, and the production of VEGF-2 polypeptides or peptides by recombinant techniques. To use them in order to do so.

Host cells are genetically treated (transduced, transformed or transfected) with the vectors of the invention, which can be, for example, cloning vectors or expression vectors. Such vectors may be in the form of, for example, plasmids, viral particles, phages, and the like. Such genetically engineered cells can be cultured in conventional nutrient media modified as appropriate to activate a promoter, select a transformant, or amplify the VEGF-2 gene of the invention. Culture conditions such as temperature, pH, etc., are those previously used with host cells selected for expression, which will be apparent to those skilled in the art.

The polynucleotide of the present invention can be used in a method for producing a polypeptide by recombinant technology. Thus, for example, the polynucleotide sequence can be included in any one of various expression vectors for expressing a particular polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences such as derivatives of SV40; Bacterial plasmids; Phage DNA; Yeast plasmids; Vectors derived from the combination of plasmid and phage DNA, vice versa, such as vaccinia, adenovirus, poultry pox virus, and pseudorabies. However, any other plasmid or vector can be used if it is replicable and viable in the host.

Appropriate DNA sequences can be inserted into the vector by a variety of methods. Generally, such DNA sequences are inserted into suitable restriction endonuclease sites by methods well known in the art. Such and other methods are considered to be within the scope of the art.

The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct mRNA synthesis. Representative examples of such promoters include the LTR or SV40 promoter, E. E. coli lac or trp, phage lambda P _L promoters, and other promoters known to modulate gene expression in prokaryotic or eukaryotic cells or their viruses may be mentioned. The expression vector also contains a ribosomal binding site and transcription terminator for initiation of translation. The vector may comprise a sequence suitable for amplifying expression.

In addition, the expression vector preferably contains one or more selectable marker genes that provide phenotypic properties for the selection of transformed host cells. Such markers include dihydrofolate reductase (DHFR) or neomycin resistance in eukaryotic cell culture, and E. coli. For culturing in E. coli and other bacteria there is tetracycline or ampicillin resistance.

By transforming the appropriate host with a vector containing the appropriate DNA sequence as mentioned above as well as the appropriate promoter or regulatory sequence, the host is able to express the protein. Representative examples of suitable hosts include: Bacterial cells such as E. coli, Salmonella typhimurium and Streptomyces; Fungal cells such as yeast; Insectoid cells such as Drosophila S2 and Spodoptera Sf9; Animal cells such as CHO, COS or Bowes melanoma; And plant cells and the like. Selection of suitable hosts is deemed to be within the scope of the art from the teachings herein.

More particularly, the present invention also encompasses recombinant constructs comprising one or more of the sequences as broadly described above. Such constructs include vectors such as plasmids or viral vectors in which the sequences of the invention have been inserted in a forward or backward direction. In a preferred aspect of this embodiment, the construct further comprises a regulatory sequence, for example comprising a promoter operably linked to the sequence. Many suitable vectors and promoters are known in the art and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (source: Qiagen), pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Source: Pharmacia). Preferred eukaryotic vectors are as follows: pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Source: Stratagene); And pSVK3, pBPV, pMSG and pSVL (Source: Pharmacia). Other suitable vectors will be readily apparent to those skilled in the art.

In addition to the use of expression vectors in practicing the present invention, the present invention further includes novel expression vectors comprising promoter and operator sequences operably linked to nucleotide sequences encoding a protein of interest. One example of such a vector is pHE4a, which is described in detail below.

As summarized in Figures 28 and 29, the components of the pHE4a vector (SEQ ID NO: 16) are 1) neomycin phosphotransferase gene, 2) as a selection marker. E. coli replication origin, 3) T5 phage promoter sequence, 4) two lac operator sequences, 5) shine-delgarno sequence, 6) lactose operon inhibitor gene (lacIq) and 7) multiple cloning site linker regions. The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). Promoter sequences and operator sequences are synthetically made. Methods of synthesizing nucleic acid sequences are well known in the art (CLONTECH 95/96 Catalog, pages 215-216, CLONTECH 1020 East Meadow Circle, Palo Alto, CA 94303). This pHE4a vector was deposited with ATCC on February 25, 1998, ATCC.

The nucleotide sequence encoding VEGF-2 (SEQ ID NO: 1) restricts this vector to NedI and XbaI, BamHI, XhoI or Asp718 and separates the larger fragment on the gel (multiple cloning site region is about 310 nucleotides) It is operatively connected to the promoter and operator. Nucleotide sequences encoding VEGF-2 with an appropriate restriction site are, for example, NedI (as 5 'primers) and XbaI, BamHI, XhoI or Asp718 (as 3' primers), according to the PCR protocol described in Example 1. It is generated using PCR primers with restriction sites for. This PCR insert is gel purified and limited to compatible enzymes. The insert and the vector are linked according to standard protocols.

As previously recognized, the pHE4a vector contains the lacIq gene. lacIq is an allele of the lacI gene that tightly regulates the lac operator (Amann, E. et al., Gene 69: 301-315 (1988); Stark, M., Gene 51: 255-267 (1987). The lacIq gene encodes an inhibitor protein that binds to the lac operator sequence and blocks transcription of the bottom (ie 3 ') sequence. However, such lacIq gene products are cleaved from the lac operator in the presence or absence of lactose or certain lactose homologues such as isopropyl B-D-thiogalactopyranoside (IPTG). Thus, VEGF-2 is not produced in appreciable amounts in uninduced host cells containing pHE4a vectors. However, inducing these host cells by adding an agent such as IPTG results in expression of the VEGF-2 coding sequence.

The promoter / operator sequence (SEQ ID NO: 17) of the pHE4a vector comprises a T5 phage promoter and two lac operator sequences. One operator is 5 'to the transcription start site and the other 3' to the same site. When these operators are present in combination with the lacIq gene product, they confer strict inhibition of the bottom sequence in the absence of lac operon inducers, eg, IPTG. Expression of operatively linked sequences located at the bottom from the lac operator can be induced by adding a lac operon inducer such as IPTG. Binding of the lac inducer to the lacIq protein initiates the transcription of the sequences from which they are released and operably linked from the lac operator sequence. Lac operon regulation of gene expression is reviewed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997) pages 802-807.

Vectors of the pHE4 series contain all components of the pHE4a vector except for the VEGF-2 coding sequence. Features of the pHE4a vector include the following: optimized synthetic T5 phage promoter, lac operator and shine-delgano sequence. In addition, these sequences are optimally spaced such that expression of the inserted gene is tightly regulated and high levels of expression occur upon induction.

Bacterial promoters known to be suitable for use in preparing the proteins of the invention include: E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, lambda PR and PL promoters and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of Raus sarcoma virus (RSV) And metallothionein promoters, such as the mouse metallothionein-I promoter.

The pHE4a vector also contains a Shine-Delgano sequence that is 5 'to the AUG start codon. The shine-delgano sequence is generally a short sequence located on top of about 10 nucleotides (ie, 5 ') from the AUG start codon. These sequences essentially direct prokaryotic ribosomes with AUG start codons.

Thus, the invention also relates to expression vectors useful for the preparation of the proteins of the invention. This aspect of the invention is illustrated by the pHE4a vector (SEQ ID NO: 16).

The promoter region can be selected from any gene of interest using a CAT (Chloramphenicol transferase) vector or other vector with a selectable marker. Two suitable vectors are PKK232-8 and PCM7. Particularly named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P _R , P _L and trp. Eukaryotic promoters include CMS Etiate Early, HSV Thymidine Kinase, Early and Late SV40, LTR from Retrovirus, and Mouse Metallothionein-I. Selection of suitable vectors and promoters is well within the scope of conventional techniques in the art.

In a further aspect, the present invention relates to a host cell containing the above-mentioned construct. The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell may be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into host cells can be performed by calcium phosphate transfection, DEAE-dextran mediated transfection, electroporation, transduction, infection, or other methods. Davis, L. , Dibner, M., Battey, I., Basic Method in Molecular Biology, (1986)].

Constructs in host cells can be used in conventional manner to produce gene products encoded by recombinant sequences. Alternatively, the polypeptides of the invention can be made synthetically by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of a suitable promoter. Cell-free translation systems can also be used to make such proteins using RNA derived from the DNA constructs of the invention. Cloning and expression vectors suitable for use with prokaryotic and eukaryotic hosts are described in Sambrook. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989), the disclosure content of which is incorporated herein by reference.

Transcription of the DNA encoding a polypeptide of the invention by higher eukaryotic cells is increased by inserting an enhancer sequence into the vector. Enhancers are typically cis-functional elements of DNA that are about 10-300 bp and act on the promoter to increase transcription. Examples include the SV40 enhancer on the end side of the origin of replication bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the end side of the origin of replication, and the adenovirus enhancer.

In general, recombinant expression vectors include: origin of replication; Selective markers that allow transformation of host cells, for example E. Coli and s. Ampicillin resistance gene of the S. cerevisiae TRP1 gene; And a promoter derived from a highly expressed gene that directs transcription of the bottom structural sequence. The promoter may be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock protein. The heterologous structural sequence is assembled in an appropriate phase with a translation initiation and termination sequence, and preferably a leader sequence that can direct the translated protein to be secreted into the periplasmic space or extracellular medium. Optionally, this heterologous sequence can encode a fusion protein comprising an N-terminal identification peptide that provides the desired properties, eg, stabilization or simplified purification of the expressed recombinant product.

Expression vectors useful for use in bacteria are constructed by inserting a structural DNA sequence encoding a protein of interest into an operative reading with a functional promoter, along with a suitable translational initiation and termination signal. The vector may comprise one or more phenotypic selectable markers and origins of replication and provide amplification in the host if necessary. Suitable prokaryotic host cells for transformation are E. coli. There are various species in E. coli, Bacillus subtilis, Svenella typhimurium, and Pseudomanas genus, Streptomyces genus and Staphylococcus genus, but others may be selected and used.

As a representative but not limited example, expression vectors useful for bacterial use may include bacterial markers of origin and selectable markers derived from commercial plasmids that include the genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Such commercially available vectors include, for example, pKK223-3 (source: Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (source: Promega Biotec, Madison, WI, USA). The pBR322 "backbone" fragment is combined with the appropriate promoter and the structural sequence to be expressed.

After transforming a suitable host strain and growing the host strain at a suitable cell density, the selected promoter is induced by a suitable method (eg temperature change or chemical induction) and the cells are cultured for an additional period of time.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.

Microbial cells used for expression of proteins can be disrupted by conventional methods, including freeze-thaw circulation, sonication, mechanical disruption, or the use of cell melters, which are well known to those skilled in the art. Can be.

In addition, various mammalian cell culture systems can be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 strain of monkey kidney fibroblasts described in Gluzman, Cell, 23: 175 (1981), and other cell lines capable of expressing a conformity vector, such as C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors include origins of replication, suitable promoters and enhancers, and essential ribosomal binding sites, polyadenylation sites, splice donor and receptor sites, transcription termination sequences, and 5 'flanking non-transcription sequences. DNA sequences derived from the SV40 viral genome, eg, the SV40 origin, early promoter, enhancer, splice and polyadenylation site, can be used to provide the necessary non-transcribed genetic elements.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also deletes or replaces endogenous genetic material (eg, VEGF-2 sequences) and / or VEGF-2 of the invention. Vertebrate origin, in particular to activate, modify and / or amplify the endogenous VEGF-2 sequences of the invention, genetically engineered to contain genetic material (e.g., heterologous promoters) that are operably linked to the sequence It encompasses primary, secondary and immortalized host cells of mammalian origin. For example, techniques known in the art can be used to operably link heterologous regulatory regions with endogenous polynucleotide sequences (e.g., encoding VEGF-2) through homologous recombination. See US Pat. No. 5,641,670. (June 24, 1997); International Publication No. WO 96/29411 (September 26, 1996); International Publication No. WO 94/12650 (August 4, 1994); Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342: 435-438 (1989); The technical content of each of these documents is incorporated herein by reference.

The host cell may be a higher eukaryotic cell, such as a mammalian cell (eg, a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell may be a prokaryotic cell, such as a bacterial cell. The host strain may be selected to modulate the expression of the inserted gene sequence or to modify and process the genetic organism in a particular manner as desired. Since expression from certain promoters can be elevated in the presence of certain inducers, expression of the genetically engineered polypeptide can be controlled. In addition, different host cells have characteristic and specific mechanisms for translation and post-translational processing and modification (glycosylation, phosphorylation, cleavage) of proteins. Appropriate cell lines can be selected to ensure the desired modification and processing of the expressed protein.

The polypeptide to ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Can be recovered and purified from recombinant cell culture. Low concentrations of calcium ions present during purification (approximately 0.1 to 5 mM) are preferred. See Price et al., J. Biol. Chem. 244: 917 (1969). Protein refolding steps can be used to complete the arrangement of mature proteins, if necessary. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.

Polypeptides of the invention are naturally purified products or by recombinant techniques from chemical synthesis processes or from prokaryotic or eukaryotic cell hosts (eg, by bacterial, yeast, higher plant, insect and mammalian cells in culture). It may be a manufactured product. Depending on the host used in the recombinant manufacturing process, the polypeptides of the invention may or may not be glycosylated with mammalian or other eukaryotic carbohydrates. Polypeptides of the invention may also comprise starting methionine amino acid residues.

In addition, the polypeptides of the invention can be chemically synthesized using techniques known in the art. See Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310: 105-111. For example, peptides corresponding to fragments of the VEGF-2 polypeptides of the invention can be synthesized by using peptide synthesizers. In addition, if desired, non-classical amino acids or chemical amino acid analogs may be introduced into the VEGF-2 polypeptide sequence as a substitution or adduct. Non-classical amino acids include, but are not limited to: D-isomers of amino acids, 2,4-diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid , g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, Cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids , And common amino acid homologues. In addition, the amino acid may be D (priority) or L (left).

The present invention is directed to, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or other cellular ligands during or after translation. VEGF-2 polypeptides that are differentially modified by, for example, ligation to and the like. Many of the chemical modifications are specific for chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH; Acetylation, formylation, oxidation, reduction; Metabolic synthesis in the presence of tunicamycin and the like, and the like.

Additional post-translational modifications encompassed by the present invention include, for example, N-linked or O-linked carbohydrate chains, N-terminus or C-terminus processing, attachment of chemical residues to amino acid backbones, N- Chemical modifications of linked or O-linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The polypeptide may also be modified with a detectable label, such as an enzyme, fluorescent, radioisotope or affinity label, which allows for detection and isolation of the protein.

Also provided herein are chemically modified derivatives of VEGF-2 that can provide additional benefits such as increased solubility, stability and circulation time of the polypeptide, or reduced immunogenicity (see US Pat. No. 4,179,337). Chemical moieties for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptide may be modified at random positions within the molecule, or may be modified at predetermined positions within the molecule and may comprise one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight and may or may not be branched. In the case of polyethylene glycol, the preferred molecular weight is from about 1 kDa to about 100 kDa (the term "about" indicates that, in the preparation of polyethylene glycol, some molecules will be more, slightly less, or less than the molecular weights mentioned in the preparation of polyethylene glycol). It is. The desired therapeutic profile (e.g., desired duration of release, effect on biological activity, if present, ease of manipulation, degree of antigenicity or lack thereof, and known polyethylene glycol to therapeutic proteins or homologues) Other effects may be used.

Polyethylene glycol molecules (or other chemical moieties) should be attached to the protein in view of its effect on the functional or antigenic domains of the protein. There are a number of methods of attachment available to those skilled in the art. See EP 0 401 384 (Coupling PEG to G-CSF), incorporated herein by reference; And Malik et al., Exp. Hematol. 20: 1028-1035 (1992) (peglycing GM-CSF with tresyl chloride has been reported). For example, polyethylene glycol can be covalently linked through amino acid residues by reactive groups such as free amino or carboxyl groups. Reactive groups are those to which activated polyethylene glycol molecules can be bound. Amino acid residues having free amino groups may include lysine residues and N-terminal amino acid residues; Amino acid residues having free carboxyl groups may include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues. Sulhydryl groups can also be used as reactive groups for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment in amino groups, for example in N-terminal or lysine groups.

Specifically desired are proteins that are chemically modified at the N-terminus. Using polyethylene glycol as an example of the composition, from various polyethylene glycols (by molecular weight, branching, etc.), the ratio of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture, the type of PEGylation reaction to be carried out, And a method for obtaining the selected N-terminally PEGylated protein. A method of obtaining an N-terminally PEGylated agent (i.e., separating the residue from other monopegylated residues as needed) is achieved by purifying the N-terminally PEGylated material from the PEGylated protein molecule population. . Selective proteins that have been chemically modified by N-terminal modifications are characterized by reductive alkylation utilizing the difference in reactivity (lysine versus N-terminus) of different types of primary amino groups that can be used for derivatization in a particular protein. Can be performed. Under appropriate reaction conditions, the carbonyl group containing polymer is used to substantially select the derivatives at the N-terminus.

The VEGF-2 polypeptides of the invention may be monomers or multimers (ie, dimers, trimers, tetramers and larger multimers). Accordingly, the present invention relates to monomers and multimers of the VEGF-2 polypeptides of the present invention, methods for their preparation, and compositions containing them (preferably pharmaceutical compositions). In certain embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In a further aspect, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

The multimers encompassed by the present invention may be homomers or heteroomers. As used herein, the term homomer refers to multimers containing only the VEGF-2 polypeptides of the invention (including VEGF-2 fragments, variants, splice variants, and fusion proteins as described herein). These homomers may contain VEGF-2 polypeptides having the same or different amino acid sequences. In certain embodiments, the homomers of the invention are multimers containing only VEGF-2 polypeptides having the same amino acid sequence. In another specific embodiment, the homomers of the invention are multimers containing VEGF-2 polypeptides having different amino acid sequences. In certain embodiments, the multimers of the invention comprise homer-dimers (eg, contain VEGF-2 polypeptides having the same or different amino acid sequences) or homo-trimers (eg, same and / or different amino acids). VEGF-2 polypeptide having a sequence). In a further aspect, the homomeric multimers of the invention are at least homo-dimer, at least homo-trimer, or at least homo- tetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to the VEGF-2 polypeptide of the invention. In certain embodiments, the multimers of the invention are hetero-dimers, hetero-trimers, or hetero- tetramers. In a further aspect, the heteromeric multimers of the invention are at least hetero-dimers, at least hetero-trimers, or at least hetero- tetramers.

Multimers of the present invention may be the result of hydrophobic, hydrophilic, ionic and / or binding linkages and / or may be indirectly linked, for example by liposome formation. Thus, in one embodiment, multimers of the invention, eg, homo-dimer or homo-trimer, are formed when the polypeptides of the invention are contacted with each other in solution. In another embodiment, the hetero-multimers of the invention, eg, hetero-trimers or hetero- tetramers, are conjugated antibodies of the invention to a polypeptide of the invention (heterologous polypeptide in the fusion protein of the invention) in solution. In contact with the sequence). In other embodiments, the multimers of the invention are formed by covalent linkages with VEGF-2 polypeptides of the invention and / or covalent linkages between the VEGF-2 polypeptides. Such covalent linkages may comprise one or more amino acid residues contained in a polypeptide sequence (eg, those contained in a polypeptide shown in SEQ ID NO: 2 or encoded by a deposited clone). In one case, such covalent linkages are crosslinks between cysteine residues located within polypeptide sequences that interact in the native (ie native) polypeptide. In another case, the covalent linkage is the result of a chemical or recombinant manipulation. Alternatively, the covalent linkage may comprise one or more amino acid residues contained in a heterologous polypeptide sequence in a VEGF-2 fusion protein. In one example, the covalent linkage may be between heterologous sequences contained in the fusion protein of the invention (see US Pat. No. 5,478,925). In certain instances, the covalent linkage is between heterologous sequences contained in the VEGF-2-Fc fusion protein of the invention (as described herein). In another specific example, the covalent linkage of a fusion protein of the invention is between another TNF family ligand / receptor member, e.g., heterologous polypeptide sequence from an osteoprotegerin, capable of forming a covalently linked multimer. [See, eg, WO 98/49305; The contents of which are incorporated herein by reference].

The multimers of the present invention can be produced using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the present invention can be chemically crosslinked using linker molecules and linker molecule length optimization techniques known in the art. See US Pat. No. 5,478,925; Which is incorporated herein by reference]. In addition, multimers of the present invention can be produced using techniques known in the art to form one or more interstitial crosslinks between cysteine residues located within the sequence of a polypeptide desired to be contained in such multimers. [US Pat. No. 5,478,925; Which is incorporated herein by reference]. In addition, polypeptides of the present invention can be modified conventionally by adding cysteine or biotin to the C-terminus or N-terminus of such polypeptides, and contain one or more of these modified polypeptides by applying techniques known in the art. Multimers can be produced. See US Pat. No. 5,478,925; Which is incorporated herein by reference]. Additionally, techniques known in the art can be applied to produce liposomes containing the polypeptide components desired to be contained in the multimers of the present invention. See US Pat. No. 5,478,925; Which is incorporated herein by reference].

Alternatively, the multimers of the present invention can be produced using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in the multimers of the invention are produced recombinantly using fusion protein techniques described herein or known in the art. See US Pat. No. 5,478,925; Which is incorporated herein by reference]. In certain instances, the polynucleotide encoding the homo-dimer of the invention links the polynucleotide sequence encoding the polypeptide of the invention to the sequence encoding the linker polypeptide, and then further from the original C-terminus to the N-terminus. Produced by linking to the synthetic polynucleotide encoding the translated product of the polypeptide in the backward direction. See US Pat. No. 5,478,925; Which is incorporated herein by reference]. In another embodiment, the recombinant polypeptides of the invention, which contain a transmembrane domain (or hydrophobic or signal peptide) by applying the recombinant techniques described herein or recombinant techniques known in the art and which can be incorporated into liposomes by membrane reconstitution techniques (See US Pat. No. 5,478,925); Which is incorporated herein by reference].

Therapeutic Uses

The VEGF-2 polypeptides of the invention are potent mitotics for vascular and lymphoid endothelial cells. As shown in Figures 12 and 13, the initial 46 amino acids minus the VEGF-2 polypeptide of SEQ ID NO: 2 are potent mitotics for vascular endothelial cells and stimulate their growth and proliferation. Northern blot assay performed on a VEGF-2 nucleic acid sequence encoding the polypeptide, which probed 20 mg of RNA from several human tissues with ³² P-VEGF-2, illustrates that the protein is actively expressed in the heart and lungs. This is further evidence of mitotic activity.

Thus, VEGF-2 or a biologically active portion thereof can be used to treat vascular injury by enhancing angiogenesis. For example, coronary bypass surgery is used to stimulate the growth of transplanted tissue. VEGF-2 or a biologically active portion thereof can also be used to promote wound healing, particularly to re-vascularize damaged tissues or to stimulate secondary blood flow during ischemia and when new capillary angiogenesis is desired. Used. VEGF-2 or a biologically active portion thereof can be used to treat significant thickness of trauma, such as skin ulcers, including pressure sores, venous ulcers and diabetic ulcers. In addition, VEGF-2 or a biologically active portion thereof can be used to treat burns and injuries of significant thickness when skin grafts or skin flaps are used to repair burns and injuries. VEGF-2 or a biologically active portion thereof may be used for use in cosmetic surgery, eg, to repair lacerations, burns or other damage. VEGF-2 can also be used to treat eye diseases as well as to promote healing of trauma and injury to the eye.

In these same contexts, VEGF-2 or a biologically active portion thereof can be used to induce the growth of damaged bone, fascia or ligament tissue. VEGF-2, or a biologically active portion thereof, can be used to regenerate its supportive tissue, including, for example, cementitious and fascia ligaments wounded by a periodontal disease or injury.

Since angiogenesis is important for cleaning trauma and preventing infection, VEGF-2 or its biologically active portion can be used in conjunction with surgery and involves repair of incisions and cuts. VEGF-2 or a biologically active portion thereof can also be used to treat abdominal trauma at high risk of infection.

VEGF-2 or a biologically active portion thereof may be used to enhance endothelialization in vascular graft surgery. In the case of vascular grafts implanted or using synthetic materials, VEGF-2 or a biologically active portion thereof may be applied to the surface or joint portion of the graft to enhance growth of vascular endothelial cells. VEGF-2 or a biologically active portion thereof can be used to repair damage to myocardial tissue due to myocardial infarction. VEGF-2 or a biologically active portion thereof can also be used to repair the cardiovascular system after ischemia. VEGF-2 or a biologically active portion thereof can be used to treat vascular tissues damaged by coronary artery disease and peripheral and CNS vascular diseases.

VEGF-2 or a biologically active portion thereof can be used to coat artificial prostheses or natural organs that are implanted into the body to minimize rejection of the implanted material and stimulate angiogenesis of the implanted material.

VEGF-2, or a biologically active portion thereof, can be used for vascular tissue repair of injury due to injury, such as that which occurs during atherosclerosis and that is required for performing balun angioplasty if the vascular tissue is damaged.

VEGF-2 or a biologically active portion thereof can be used to treat peripheral arterial disease. Thus, in a further aspect, a method of using a VEGF-2 polypeptide to treat peripheral arterial disease is provided. Preferably, the VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating peripheral arterial disease. Suitable doses, formulations and routes of administration are described below.

VEGF-2, or a biologically active portion thereof, may enhance endothelial function of lymphoid tissues and blood vessels, such as the loss of lymphatic vessels, occlusion of lymphatic vessels, and lymphangiomas. VEGF-2 can also be used to stimulate lymphocyte production.

VEGF-2 or a biologically active portion thereof can be used to treat hemangiomas in newborns. Thus, in a further aspect, a method of using a VEGF-2 polypeptide to treat an hemangioma in a newborn is provided. Preferably, the VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating hemangioma in a newborn. Suitable doses, formulations and routes of administration are described below.

VEGF-2 or a biologically active portion thereof can be used to treat abnormal retinal development in immature newborns. Thus, in a further aspect, a method of using a VEGF-2 polypeptide to treat abnormal retinal development in immature newborns is provided. Preferably, the VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating abnormal retinal development in immature newborns. Suitable doses, formulations and routes of administration are described below.

VEGF-2 or a biologically active portion thereof can be used to treat primary (idiopathic) lymphedema, including Milroy's disease and premature lymphoma. Thus, in a further aspect, a method of using a VEGF-2 polypeptide to treat primary (idiopathic) lymphedema, including Miloy's and precocious lymphedema, is provided. Preferably, the VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating primary (idiopathic) lymphedema, including Miloy's disease and premature lymphoma. Suitable doses, formulations and routes of administration are described below.

VEGF-2 or its biologically active portion can be used to (i) remove lymph nodes and lymphatic vessels, (ii) treat radiotherapy and surgery in cancer treatment, and (iii) treat secondary (occlusive) lifespan resulting from injury and infection. have. Thus, in a further aspect, VEGF-2 to treat (i) lymph node and lymphatic vessel removal, (ii) radiation therapy and surgery in cancer treatment, and (iii) the secondary (occlusive) lifespan resulting from injury and infection. Methods of using a polypeptide are provided. Preferably, the VEGF-2 polypeptide is administered to an individual for (i) lymph node and lymphatic vessel removal, (ii) radiation therapy and surgery in cancer treatment, and (iii) secondary (occlusive) lifespan resulting from injury and infection. do. Suitable doses, formulations and routes of administration are described below.

VEGF-2 or a biologically active portion thereof can be used to treat Kaposi's sarcoma. Thus, in a further aspect, a method of using a VEGF-2 polypeptide to treat Kaposi's sarcoma is provided. Preferably, the VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating Kaposi's sarcoma. Suitable doses, formulations and routes of administration are described below.

VEGF-2 antagonists can be used to treat cancer by inhibiting angiogenesis necessary to support cancer and tumor growth.

Cardiovascular disorders

We found that VEGF-2 stimulates vascular endothelial cell growth, stimulates endothelial cell migration, stimulates angiogenesis in the CAM assay, lowers blood pressure in spontaneous hypertensive rats and blood flow to ischemic limbs in rabbits. It was found to increase the. Thus, VEGF-2 polypeptides or polynucleotides encoding VEGF-2 can be used to treat peripheral arterial diseases such as cardiovascular disorders including lichen ischemia.

Cardiovascular disorders include cardiovascular abnormalities such as sinus aneurysms, arteriovenous varices, cardiac arteriovenous malformations, congenital heart defects, pulmonary artery obstruction, and Scimitar syndrome. Congenital heart defects include aortic stenosis, triangulation, coronary anomaly, cruciate heart, right heart, open arterial canal, Ebstein's abnormality, Eismenger's syndrome, attenuated right atrial syndrome, left cardiacism, palosing, large vessels Anterior chamber, double vented right ventricle, tricuspid occlusion, persistent arterial and cardiac septal defects, such as aortic pulmonary septal defect, endocardial cussion defect, Lutembacher, palosing, ventricular cardiac septal defect This includes.

Cardiovascular disorders also include heart diseases such as arrhythmia, carcinoid heart disease, high cardiac output, low cardiac output, cardiac piesm, endocarditis (including bacteria), heart flow, cardiac arrest, congestive heart failure, congestion Sexual cardiomyopathy, paroxysmal respiratory distress, heart edema, cardiac hypertrophy, cognitive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarct heart rupture, ventricular septal rupture, heart valve disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (stenosis) And tuberculosis emphysema, post-pericardial incision-post-syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy intoxication, simitar syndrome, cardiovascular syphilis and cardiovascular tuberculosis.

Arrhythmia includes homoarrhythmia, atrial fibrillation, atrial flutter, bradycardia, extracorporeal contraction, Adams-Stokes syndrome, angular block, homoblock, intestinal QT syndrome, collateral contraction, low-garnon-levin syndrome, hemp Heim-type pre-excitation syndrome, Wolf-Parkinson-Vite syndrome, diseased same-sex syndrome, anemia and ventricular fibrillation. Anemias include paroxysmal anemia, loss of anemia, accelerated ventricular intrinsic rhythm, atrioventricular nodule recurrent anemia, ectopic atrial anemia, unilateral neuropathy, homosexual nodular retinopathy, gay dystonia, tor Torsades de Points and ventricular anemia.

Cardiac valve diseases include aortic valve insufficiency, aortic valve stenosis, heart murmur, aortic valve deprivation, mitral pantalum, tricuspid valve, mitral insufficiency, mitral valve stenosis, pulmonary artery obstruction, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid insufficiency, tricuspid insufficiency and tricuspid stenosis do.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subaortic valve stenosis, subpulmonary valve stenosis, limited cardiomyopathy, Kagas cardiomyopathy, endocardial fibroelasticity, myocardial fibrosis, Cairns syndrome, myocardial reperfusion injury, and myocarditis do.

Myocardial ischemia includes coronary diseases such as angina pectoris, coronary artery, coronary atherosclerosis, coronary thrombosis, coronary angiospasm, myocardial infarction and myocardial fainting.

Cardiovascular disorders also include vascular diseases, such as aneurysms, angioplasty, angiomatosis, bacterial angiomatosis, Hippel-Lindau disease, Kippel-Trenaunai-Weber syndrome, Steg-Weber syndrome, angioedema , Aortic disease, arthritis of Takayasu, aorticitis, Lerich's syndrome, arterial obstructive disease, arteritis, spheroiditis, nodular polyarteritis, cardiovascular disorders, diabetic angiopathy, diabetic retinopathy, embolism, thrombosis, dermatitis, Hemorrhoids, hepatic vein-obstructive disease, hypertension, hypotension, ischemia, peripheral vascular disease, pulmonary vein-obstructive disease, Roynaud's disease, CREST syndrome, retinal vein occlusion, simitar syndrome, superior venous vena cava syndrome, capillary dilator, atta Sia capillary dilator, hereditary hemorrhagic capillary dilator, venous arthroscopy, venous venous vein, venous venous ulcer, vasculitis and venous insufficiency.

Aneurysms include dissociative aneurysms, gastric aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms and iliac aneurysms.

Arterial obstructive diseases include atherosclerosis, intermittent claudication, carotenoid stenosis, myofiber dysplasia, mesenteric vascular occlusion, moyamoya disease, renal artery occlusion, retinal artery occlusion, and thrombovascular occlusion.

Cerebrovascular disorders include carotened arterial disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral oxygen deficiency, cerebral atherosclerosis, cerebral arterial dysplasia, cerebral artery disease, cerebral embolism and thrombosis, carotened arterial thrombosis, homothrombosis, Valenberg syndrome, Cerebral hemorrhage, epidural hematoma, subdural hematoma, subdural hemorrhage, cerebral infarction, cerebral ischemia (including transient), Tinun's Still syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine and basal spinal insufficiency Included.

Embolisms include air embolism, amniotic embolism, cholesterol embolism, blue earth syndrome, fat embolism, pulmonary embolism and thromboembolism. Thrombosis includes coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotened arterial thrombosis, homosexual thrombosis, Wallenberg syndrome and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndrome, anterior compartment syndrome, myocardial ischemia, reperfusion injury, and peripheral ischemia. Vasculitis includes aorticitis, arteritis, Bensham syndrome, Jerg-Strauss syndrome, mucosal skin lymph node syndrome, thrombophlebitis obstruction, irritable phlebitis, Schoenlein-Henno purpura, allergic subcutaneous phlebitis and granulitis of Wegener.

VEGF-2 polypeptides or polynucleotides are particularly useful for the treatment of severe lichen ischemia and coronary disease. As shown in FIG. 18, administration of the VEGF-2 polynucleotide and polypeptide to the experimentally induced ischemic rabbit Fuji restored blood pressure ratio, blood flow, angiographic score and capillary density.

VEGF-2 polypeptides are injected directly into the delivery site with a needle, intravenous injection, topical administration, catheter injection, bioplastic syringe, particle promoter, gelfoam sponge depots, other commercial depot materials, osmotic pumps, oral or suppository solid pharmaceuticals The composition can be administered using methods known in the art including, but not limited to, gradient injection or topical administration during surgery, aerosol delivery. Such methods are known in the art. The VEGF-2 polypeptide may be administered as part of a pharmaceutical composition, as described in more detail below. Methods of carrying VEGF-2 polynucleotides are described in more detail below.

Gene therapy method

Another aspect of the invention relates to gene therapy methods for treating disorders, diseases and symptoms. Such gene therapy methods relate to introducing nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into certain animals to achieve expression of the VEGF-2 polypeptide of the invention. This method requires a polynucleotide encoding a VEGF-2 polypeptide operably linked to a promoter, and other genetic elements essential for expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art (see, eg, WO 90/11092, incorporated herein by reference).

Thus, for example, a patient's cells may be genetically engineered in vitro with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a VEGF-2 polynucleotide, and then such genetically engineered cells may be engineered. It may be provided to a patient to be treated with the polypeptide. Such methods are well known in the art. See Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L. et al., Human Gene Therapy 7: 1-10 (1996); Santodonato, L., et al., Gene Therapy 4: 1246-1255 (1997); and Zhang, J. F. et al., Cancer Gene Therapy 3: 31-38 (1996); These are incorporated herein by reference. In one embodiment, the genetically treated cells are arterial cells. The arterial cells can be reintroduced into the patient by directly injecting into the artery, the tissue surrounding the artery, or by catheter injection.

As will be discussed in more detail below, the VEGF-2 polynucleotide construct may be used to deliver an injectable substance to cells of an animal, such as the epilepsy region of tissue (heart, muscle, skin, lung, liver, etc.). It can be delivered by injection into. The VEGF-2 polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the VEGF-2 polynucleotide is delivered as an exposed polynucleotide. The term “exposed” polynucleotide, DNA, or RNA refers to any transport vehicle that acts to assist, enhance or promote the introduction into cells, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents, and the like. Refers to sequences that do not have. However, VEGF-2 polynucleotides can be delivered in liposome formulations, lipofectin formulations, and the like, and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference.

The VEGF-2 polynucleotide vector constructs used in gene therapy methods are preferably constructs that do not integrate into the host genome or contain no sequences that permit replication. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (Source: Stratagene); pSVK3, pBPV, pMSG and pSVL (Source: Pharmacia); And pEF1 / V5, pcDNA3.1 and pRc / CMV2 (Invitrogen). Other suitable vectors will be readily apparent to those skilled in the art.

Any strong promoter known to those skilled in the art can be used to drive the expression of VEGF-2 DNA. Suitable promoters include adenoviral promoters such as adenoviral major late promoters; Heterologous promoters such as cytomegalovirus (CMV) promoters; Respiratory syncytial virus (RSV) promoter; Inducible promoters such as MMT promoter and metallothionein promoter; Heat shock promoter; Albumin promoter; ApoAI promoter; Human globin promoter; Viral thymidine kinase promoters such as the simple herpes thymidine kinase promoter; Retroviral LTR; b-actin promoter; And human growth hormone promoters. The promoter may be the original promoter for VEGF-2.

Unlike other gene therapy techniques, one major advantage of introducing exposed nucleic acid sequences into target cells is the temporaryity of polynucleotide synthesis in the cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to produce the desired polypeptide for a period of up to six months.

VEGF-2 polynucleotide constructs include muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testes, ovaries, uterus, It can be transported to the interstitial space of tissues in animals, including the rectum, nervous system, eyes, gland and connective tissue. The interstitial spaces of such tissues include mucopolysaccharide matrices of intercellular fluid, elastic fibers in blood vessel or chamber walls, collagen fibers of fibrous tissue, or cattle in connective tissue or bone covering muscle cells, among the reticular fibers of organ tissue. It contains the same matrix within. Similarly, lymphatic fluid of spatial and lymphatic channels occupied by circulating plasma is included. Delivery of muscle tissue to the interstitial space is preferred for the reasons discussed below. They can be conveniently delivered by injection into tissues comprising these cells. They are preferably achieved in cells that are transported and expressed in persistent non-differentiating cells to be differentiated, but which are not differentiated or less fully differentiated, for example, liver cells or blood fibroblasts of blood. Can be. Muscle cells in vivo are particularly qualified in terms of their ability to absorb and express polynucleotides.

For exposed acid sequence injections, an effective dosage of DNA or RNA will range from about 0.05 to about 50 mg / kg body weight. The dosage will preferably be about 0.005 to about 20 mg / kg body weight, more preferably about 0.05 to about 5 mg / kg body weight. Of course, as will be appreciated by those skilled in the art, such dosage will vary depending on the tissue site being injected. Appropriate and effective dosages of nucleic acid sequences can be readily determined by those skilled in the art and can depend on the disease to be treated and the route of administration.

Preferred routes of administration are parenteral injection routes into the interstitial space of tissues. However, other parenteral routes, such as lung or bronchial tissue, throat or nasal mucosa, can also be used. In addition, the exposed VEGF-2 DNA construct can be transported to the artery during angioplasty by the catheter used in the procedure.

The exposed polynucleotide can be delivered by methods known in the art, including but not limited to, direct injection with a needle into the delivery site, intravenous injection, topical administration, catheter injection and so-called "gene guns". have. These methods of transport are known in the art.

As demonstrated by Example 18, when the exposed VEGF-2 nucleic acid sequence is administered in vivo, the VEGF-2 polypeptide is successfully expressed in the femoral vein of the rabbit.

The construct may also be delivered in a delivery vehicle, such as viral sequence, viral particles, liposome formulation, lipofectin, precipitant, and the like. Such methods of transport are known in the art.

In certain embodiments, the VEGF-2 polynucleotide construct forms a complex in the liposome formulation. Liposomal preparations for use in the present invention include cationic (positive charges), anionic (negative charges) and neutral preparations. However, cationic agents are particularly preferred because firmly charged complexes can form between cationic liposomes and polyanionic nucleic acids. Cationic liposomes are functional forms of plasmid DNA [Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416, incorporated herein by reference; mRNA [Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86: 6077-6081, incorporated herein by reference; And purified transcription factors [Debs et al., J. Biol. Chem. (1990) 265: 10189-10192, incorporated herein by reference.

Cationic liposomes are readily available. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposomes are particularly useful and are commercially available under the trade name Lipofectin [Source: GIBCO BRL, Grand Island, NY; Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416, incorporated herein by reference. Other commercially available liposomes include Transfectase (DDAB / DOPE) and DOTAP / DOPE (Boehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. For example, specifications for the synthesis of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane) liposomes are described in PCT Publication No. WO 90/11092, incorporated herein by reference. ). Methods for preparing DOTMA liposomes are described in Felgener et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, incorporated herein by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are described, for example, by Avanti Polar Lipids; Birmingham, Ala] or readily available using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphosphatidyl ethanolamine (DOPE). These materials can also be mixed with DOTMA and DOTAP starting materials in suitable proportions. Methods for preparing liposomes using these materials are well known in the art.

For example, commercially available dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG) and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to add convenient liposomes with or without adding cholesterol. It can manufacture. Thus, for example, DOPG / DOPC endoplasmic reticulum can be prepared by drying 50 mg each of DOPG and DOPC into an ultrasonic vial under a nitrogen gas stream. The sample is left under vacuum pump overnight and hydrated with deionized water the next day. The sample is then sonicated for 2 hours in a capped vial using a Heat Systems Model 350 sonicator equipped with a cup (bath type) probe inverted under maximum setting, during which the bath is circulated to 15EC. Alternatively, the negatively charged vesicles can be prepared without sonication to produce multilayer thin film vesicles or to produce monolayer thin films of discrete sizes by extraction through nucleophor membranes. Other methods are known and available to those skilled in the art.

Liposomes may comprise multilayer thin film vesicles (MLV), small monolayer thin film vesicles (SUV) or large monolayer thin film vesicles (LUV), with SUV being preferred. Various liposome-nucleic acid complexes are prepared using methods known in the art (Straubinger et al., Methods of Immunology (1983), 101: 512-527, incorporated herein by reference). For example, MLV-containing nucleic acids can be prepared by attaching a phospholipid of a thin film on a glass tube wall and then hydrating it with a solution of the material to be encapsulated. SUVs are prepared by extending sonication of the MLV to provide a homogeneous population of monolayer thin film liposomes. The material to be captured is added to a suspension of the performed MLV and then sonicated. If liposomes containing cationic lipids are used, the dried lipid film is resuspended in a suitable solution, such as sterile water, or an isotonic buffer solution, such as 10 mM Tris / NaCl, sonicated, and then liposomes performed as such. Mix directly with DNA. Liposomes and DNA form very stable complexes due to their binding to positively charged liposomes for cationic DNA. SUVs are used with small nucleic acid fragments. LUVs are prepared by a number of methods well known in the art. Commonly used methods include Ca ²⁺ -EDTA chelation [Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394: 483; Wilson et al., Cell (1979) 17:77; Ether injection [Deamer, D. and Bangham, A., Biochem. Biophys. Acta. (1976) 443: 629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76: 836; Fraleyt et al., Proc. Natl. Acad. Sci. USA (1979) 76: 3348; Detergent dialysis [Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA (1979) 76: 145; And reversed phase evaporation (REV) [Frayley et al., J. Biol. Chem. (1980) 255: 10431; Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75: 145; Schaefer-Ridder et al., Science (1982) 215: 166; Incorporated herein by reference.

In general, the ratio of DNA to liposomes will be from about 10: 1 to about 1:10. Preferably, this ratio will be about 5: 1 to about 1: 5. More preferably, the ratio will be about 3: 1 to about 1: 3, even more preferably about 1: 1.

U.S. Pat. No. 5,676,954, which is incorporated herein by reference, reports the injection of genetic material into a mouse complexed with a cationic liposome carrier. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055 and WO 94/9469 (incorporated herein by reference). Cationic lipids are provided for use in transfecting DNA into cells and mammals. U.S. Pat.Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055 and WO 94/9469 (incorporated herein by reference) describe methods for delivering DNA-cationic lipid complexes to mammals. have.

In certain embodiments, the cells are genetically engineered ex vivo or in vivo using retroviral particles containing RNA comprising a sequence encoding VEGF-2. Retroviruses capable of inducing retroviral plasmid vectors include, but are not limited to, Moloney Murine Leukemia Virus, Spleen Necrosis Virus, Raus Sarcoma Virus, Harvey Sarcoma Virus, Avian Leukemia Virus, Gibbon Ape Leukemia Virus, Human Immunodeficiency Virus , Adenoviruses, myeloproliferative sarcoma viruses, and breast tumor viruses.

Packaging cell lines are transduced using retroviral plasmid vectors to form producer cell lines. Examples of packaging cells that can be transfected include PE501, PA317, R-2, R as described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated by reference in its entirety. -AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP + E-86, GP + envAm12, and DNA cell lines, but are not limited thereto. The vector transduces the packaging cells by any means known in the art. Such means include, but are not limited to, electrophoresis, the use of liposomes, and CaPO ₄ precipitation. In one alternative embodiment, the retroviral plasmid vector can be encapsulated into liposomes or coupled with a lipid and then administered to a host.

Producer cell lines produce infectious retroviral vector particles comprising polynucleotides encoding VEGF-2. The retroviral vector particles are then used to transduce eukaryotic cells in vitro or in vivo. Such transduced eukaryotic cells will express VEGF-2.

In certain other embodiments, the cells are genetically engineered ex vivo or in vivo using VEGF-2 polynucleotides contained in adenovirus vectors. Adenoviruses can be engineered to inactivate in terms of their ability to encode and express VEGF-2 and at the same time replicate the normal soluble viral cycle. Adenovirus expression is achieved without the integration of viral DNA into the host cell chromosome, thereby eliminating concerns about mutant insertion. Moreover, adenoviruses have been used for a long time as live intestinal vaccines with excellent stability profiles (see Schwatrz, A. R. et al. (1974) Am. Rev. Respir. Dis. 109: 233-238. Finally, adenovirus mediated gene transfer has been demonstrated in a number of cases, including alpha-1-antitrypsin and CFTR to cotton rats' lungs. Rosenfeld, M.A., et al. (1991) Science 252: 431-434; Rosenfeld et al., (1992) Cell 68: 143-155. In addition, extensive studies to demonstrate that adenovirus is a pathogen in human cancer are consistently negative [Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76: 6606.

Suitable adenovirus vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3: 499-503 (1993); Rosenfeld et al., Cell 68: 143-155 (1992); Engelhardt et al., Human Genet. Ther. 4: 759-769 (1993); Yang et al., Nature Genet. 7: 362-369 (1994); Wilson et al., Nature 365: 691-692 (1993); and US Pat. No. 5,652,224; Incorporated herein by reference. For example, adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the El region of the adenovirus and continuously express Ela and Elb that complement the defective adenovirus by providing the product of the gene deleted from the vector. In addition to Ad2, other variants of adenoviruses such as Ad3, Ad5 and Ad7 are also useful in the present invention.

Preferably, the adenovirus used in the present invention is replication deficient. Replication deficient adenoviruses require the help of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus can infect cells and can express a promoter, eg, a polynucleotide of interest that is operably linked to the HARP promoter of the invention, but cannot be replicated in most cells. Replication deficient adenovirus may be deleted with one or more of some or all of the following genes: Ela, Elb, E3, E4, E2a or L1 to L5.

In certain other embodiments, adeno-associated virus (AAV) is used to genetically treat cells ex vivo or in vivo. AAV is a naturally defective virus that needs helper viruses to produce infectious particles. Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158: 97 (1992). It is also one of several viruses that can integrate its DNA into non-differentiating cells. Vectors containing as few as 300 base pairs of AAV can be packaged and integrated, but the space for exogenous DNA is limited to about 4.5 kb. Methods of making and using such AAVs are known in the art. See, for example, US Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377. number].

For example, AAV vectors suitable for use in the present invention will include all of the sequences necessary for DNA replication, encapsidation, and host cell integration. Such VEGF-2 polynucleotide constructs are described, for example, in Sambrook. Insert into AAV vectors using standard cloning methods, as described in et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells infected with the helper virus using all standard techniques including lipofectin, electroporation, calcium phosphate precipitation and the like. Suitable helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses or herpes viruses. When packaging cells are transfected and infected, they will produce infectious AAV viral particles containing the VEGF-2 polynucleotide construct. These viral particles are then used to transduce eukaryotic cells ex vivo or in vivo. Such transduced cells will contain the VEGF-2 polynucleotide construct integrated into its genome and express VEGF-2.

Another gene therapy method involves operably linking a heterologous regulatory region with an endogenous polynucleotide sequence (eg, encoding VEGF-2) via homologous recombination. US Pat. No. 5,641,670 (June 24, 1997) ); International Publication WO 96/29411 (September 26, 1996); International Publication WO 94/12650 (August 4, 1994); Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989). Such methods include activating genes that are present in the target cell but are not normally expressed in the cell or are expressed at levels below the desired level.

Using standard techniques known in the art, polynucleotide constructs containing a promoter with a targeting sequence flanking the promoter are made. Suitable promoters are as described herein. The targeting sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination of the promoter-targeting sequence with the endogenous sequence. Since the targeting sequence is sufficiently near the 5 'end of the VEGF-2 target endogenous polynucleotide sequence, such a promoter will be operably linked to the endogenous sequence upon homologous recombination.

Promoter and targeting sequences can be amplified using PCR. Preferably, such amplified promoters contain separate restriction enzyme sites on the 5 'and 3' ends. Preferably, the 3 'end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second targeting sequence is the same restriction enzyme site as the 3' end of the amplified promoter It contains. The promoter and targeting sequences thus amplified are digested and linked together.

Promoter-targeting sequence constructs are delivered to cells as exposed polynucleotides or mixed with transfection promoters, such as liposomes, viral sequences, viral particles, complete viruses, lipofectin, precipitants, etc., as described in more detail above. do. P promoter-targeting sequences can be delivered by methods including direct injection with a needle, intravenous injection, topical administration, catheter injection, particle promoter, and the like. This method is described in more detail below.

Promoter-targeting sequence constructs are taken up by cells. Homologous recombination occurs between the construct and the endogenous sequence, eg, endogenous VEGF-2 sequences are left under the control of a promoter. The promoter then drives the expression of endogenous VEGF-2 sequences.

Polynucleotides encoding VEGF-2 can be administered with other polynucleotides encoding other angiogenic proteins. Angiogenic proteins include acidic and basic fibroblast growth factor, VEGF-1, epidermal growth factor alpha and beta, platelet-induced endothelial growth factor, platelet-induced growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin Like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte / macrophage colony stimulating factor and nitric oxide synthase.

Preferably, the polynucleotide expressing VEGF-2 contains a secretory signal sequence that promotes secretion of the protein. Typically, the signal sequence is located in the coding region of the polynucleotide to be expressed towards or at the 5 'end of the coding region. This signal sequence may be homologous or heterologous to the polynucleotide of interest and homologous or heterologous to the cell to be transfected. Additionally, this signal sequence can be chemically synthesized using methods known in the art.

Any mode of administration of the above-mentioned polynucleotide constructs can be used as long as such a way that one or more molecules are expressed in sufficient amount to provide a therapeutic effect. These include direct needle injections, systemic injections, catheter injections, bioplastic syringes, particle accelerators (eg "gene guns"), gelfoam sponge depots, other commercial depot materials, osmotic pumps (eg alza minipumps), oral or suppository solids. (Tablets or pills) pharmaceutical formulations, and gradient injection or topical administration during surgery. For example, direct injection of an exposed calcium phosphate-precipitated plasmid into the rat liver and rat spleen, or direct injection of a protein coated plasmid into the portal vein, resulted in gene expression in rat liver. Kaneda et al. , Science 243: 375 (1989).

Preferred topical methods of administration are by direct injection. Preferably, the recombinant molecules of the present invention complexed with the delivery vehicle are administered by direct injection into the artery or localized in the arterial area. Administration of a composition localized in an arterial region refers to the injection of several centimeters of the composition, preferably millimeters, into the artery.

Another topical administration method is to contact the polynucleotide construct of the present invention at or near the surgical wound site. For example, the patient may undergo surgery and coat the polynucleotide construct on the tissue surface inside the wound or inject the construct into the wound internal tissue area.

Therapeutic compositions useful for systemic administration include the recombinant molecules of the invention in complex with the targeted delivery vehicle of the invention. Suitable delivery vehicles for use in systemic administration include ligands for targeting such vehicles to specific sites.

Preferred systemic methods of administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injection can be performed using standard methods in the art. Aerosol delivery can be performed using standard methods in the art. See Stribling et al., Proc. Natl. Acad. Sci. USA 189: 11277-11281, 1992; Incorporated herein by reference. Oral delivery can be performed by forming a polynucleotide construct of the present invention in complex with a carrier capable of sustained degradation by degradative enzymes on an animal. Examples of such carriers include plastic capsules and tablets as known in the art. Local delivery can be accomplished by mixing the polynucleotide constructs of the invention with a lipophilic reagent (eg, DMSO) that can be passed into the skin.

Determining the effective amount of a substance to be delivered will depend on a number of factors, including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the exact condition and severity thereof requiring treatment, and the route of administration. Can be. The number of treatments depends on a number of factors such as the health and history of the subject as well as the amount of polynucleotide construct administered per batch. The exact amount, frequency of administration, and time of administration will be determined by the attending physician or veterinarian.

The therapeutic compositions of the invention can be administered to all animals, preferably mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cattle, horses and pigs, with human beings being particularly preferred.

Utilization of Nucleic Acids

VEGF-2 nucleic acid sequences and VEGF-2 polypeptides can be used for in vitro purposes related to scientific research, DNA synthesis, and construction of DNA vectors, and to prepare diagnostic and therapeutic agents for treating human diseases. For example, VEGF-2 can be used to culture vascular endothelial cells in vitro, where it is added to the conditioning medium at a concentration of 10 pg / ml to 10 ng / ml.

Fragments of the full-length VEGF-2 gene can be used as hybridization probes for the cDNA library to isolate other genes with sequences similar to those genes or with similar biological activity. Probes of this type generally have 50 or more base pairs, but they can have a larger number of bases. The probe can also be used to identify cDNA clones corresponding to full length transcripts and genomic clone (s) containing the complete VEGF-2 gene, including regulatory and promoter regions, exons and introns. One example of the screen involves separating the coding region of the VEGF-2 gene by using a DNA sequence known to synthesize oligonucleotide probes. Labeled oligonucleotides having sequences complementary to the sequences of the genes of the invention are used to screen libraries of human cDNA, genomic DNA or mRNA to determine members of the library that hybridize with the probe.

The present invention provides a method for identifying the VEGF-2 receptor. Genes encoding such receptors can be identified by a number of methods known to those skilled in the art, such as ligand panning and FACS classification. Coligan, et al., Current Protocols in Immun ., 1 (2), Chapter 5, (1991)]. Preferably, expression cloning prepared from cells reactive to VEGF-2 with polyadenylation RNA is used, and the cDNA library generated from such RNA is partitioned into pools and used to react with VEGF-2. Transfect free COS cells or other cells. Transfected cells grown on glass slides are exposed to labeled VEGF-2. VEGF-2 can be labeled in a variety of ways, including iodide or incorporation of a recognition site for site specific protein kinases. After fixation and incubation, the slides are analyzed using autoradiographing. Positive pools are identified, sub-pools are prepared, and retransfected using repeated sub-pooling and rescreening methods to yield a single clone that finally encodes the putative receptor.

As another approach to receptor identification, labeled VEGF-2 can be photoaffinityly linked to an extract preparation that expresses a cell membrane or receptor molecule. The crosslinked material is split by PAGE and exposed to X-ray film. The labeled complex containing VEGF-2 is cleaved, split into peptide fragments and protein microsequenced. Amino acid sequences obtained from microsequencing can be used to design a set of denatured oligonucleotide probes for screening cDNA libraries to identify genes encoding putative receptors.

VEGF-2 agonists and antagonists

The invention also relates to methods of screening compounds for identifying VEGF-2 agonists or VEGF-2 antagonists. One example of this method takes advantage of the ability of VEGF-2 to significantly stimulate the proliferation of human endothelial cells in the presence of auxiliary comitogen Con A. Endothelial cells are obtained and cultured in 96-well flat culture plates (Costar, Cambridge, Mass.), In reaction mixtures supplemented with Con-A (Calbiochem, La Jolla, Calif.). Con-A, the polypeptide of the invention and the compound to be screened are added. After incubation at 37 ° C., the cultures were pulsed with ¹ FCi of [ ³ H] thymidine (5Ci / mmol; 1Ci = 37BGq; NEN) for a time sufficient to incorporate ³ [H] and a glass fiber filter (manufactured by Harvested on Cambridge Technology, Watertown, Mass. The average ³ [H] thymidine incorporation (cpm) of triplicate cultures is measured using a liquid scintillation counter (Beckman Instruments, Irvine, Calif.). Significant ³ [H] thymidine incorporation indicates stimulation of endothelial cell proliferation compared to control assays in which the compound is excluded.

To assay for antagonists, the assays mentioned above are performed and the ability of the compound to inhibit ³ [H] thymidine incorporation in the presence of VEGF-2 indicates that the compound is an antagonist for VEGF-2. Alternatively, VEGF-2 antagonists can be detected by binding VEGF-2 and potent antagonists with membrane bound VEGF-2 receptors or recombinant receptors under conditions suitable for competitive inhibition assays. VEGF-2 can be labeled with radiation or the like, such that the number of VEGF-2 molecules bound to the receptor can determine the efficacy of the potent antagonist.

Alternatively, after interacting VEGF-2 and the receptor, the response of a known second messenger system is measured and compared in the presence or absence of the compound. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channel or phosphinositide hydrolysis. In another method, a mammalian cell or membrane preparation that expresses a VEGF-2 receptor is incubated with labeled VEGF-2 in the presence of the compound. The ability of the compound to enhance or block the interaction can then be measured.

Efficacy VEGF-2 antagonists include antibodies or in some cases oligonucleotides, which effectively destroy VEGF-2 function by binding to a polypeptide of the invention. Alternatively, an effective antagonist may be a closely related protein that binds to the VEGF-2 receptor, but because they are inactive forms of the polypeptide, they inhibit the action of VEGF-2. Examples of these antagonists are negative dominant mutants of VEGF-2 polypeptides, for example, one chain of the hetero-dimeric form of VEGF-2 may be dominant and mutated so that biological activity is not retained. One example of a negative dominant mutant includes a truncated version of dimeric VEGF-2 that can interact with another dimer to form wild type VEGF-2, but the resulting homo-dimer is inactive and characterized. No VEGF-2 activity.

Another potent VEGI antagonist is an antisense construct prepared using antisense technology. Antisense technology is used to regulate gene expression via antisense DNA or RNA or to regulate gene expression through triple-helix formation. All of these methods are based on binding polynucleotides to DNA or RNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a mature polypeptide of the invention can be used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to gene regions involved in transcription, thereby inhibiting the transcription and production of VEGF-2 [triple helix-Lee et al., Nucleic. Acids Research, 6: 3073 (1979); Cooney et al, Science, 241: 456 (1988); And Dervan et al., Science, 251: 1360 (1991). Antisense RNA oligonucleotides hybridize with mRNA in vivo and block the mRNA molecule from being translated into VEGF-2 polypeptides [antisense-Okano, J. Neurochem., 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)]. It also carries the above-mentioned oligonucleotides into cells, whereby antisense RNA or DNA is expressed in vivo to inhibit the production of VEGF-2. Do it.

Efficacy VEGF-2 antagonists also include small molecules that bind to and occupy the active site of the polypeptide, thereby making the catalytic site inaccessible to the substrate and inhibiting normal biological activity. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules.

The antagonist can be used to limit the angiogenesis required for solid tumor metastasis. Identification of VEGF-2 can be used to generate specific inhibitors of vascular endothelial growth factor. Since angiogenesis and angiogenesis are essential steps for solid tumor growth, inhibiting angiogenic activity of vascular endothelial growth factor is very useful for further growth prevention, delay or even inhibition of solid tumors. Although the level of expression of VEGF-2 is significantly lower in normal tissues, including breasts, it has been found that it can be expressed at moderate levels in two or more breast tumor cell lines derived from malignant tumors. Thus, it is possible for VEGF-2 to be involved in tumor angiogenesis and growth.

Gliomas are a specific type of neoplasia that can be treated using the antagonists of the invention.

The antagonist may also be used to treat chronic inflammation caused by increased vascular permeability. In addition to these disorders, the antagonists can be used to treat retinopathy associated with diabetes, rheumatoid arthritis and psoriasis.

Such antagonists can be used in the compositions, for example, with pharmaceutically vanity carriers as described below.

Pharmaceutical composition

The VEGF-2 polypeptides, polynucleotides, and agonists and antagonists of the invention can be used in combination with suitable pharmaceutical carriers. Such compositions comprise a therapeutically effective amount of the polypeptide, or agonist or antagonist, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should be suitable for the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the invention. These containers are affixed with a notice in the form prepared by a governmental agency that regulates the manufacture, use or sale of a pharmaceutical or biological product, which reflects the approval of the manufacture, use or sale of the drug for human administration by the governmental agency. have. In addition, the pharmaceutical compositions of the present invention can be used with other therapeutic compounds.

The pharmaceutical compositions of the invention can be administered in a convenient manner, eg, by the topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes. The pharmaceutical composition is administered in an amount effective to treat and / or prevent specific indications. In general, they are administered in an amount of at least about 10 g / kg body weight and in most cases they will be administered in an amount of no greater than about 8 mg / kg body weight per day. In most cases, the dosage is about 10 g / kg body weight to about 1 mg / kg body weight, taking into account the route of administration, symptoms, and the like.

VEGF-2 polypeptides, and agonists or antagonists that are polypeptides, can be utilized in accordance with the present invention by expressing such polypeptides in vivo, which is often referred to as "gene therapy" described above.

Thus, for example, cells such as bone marrow cells can be genetically treated with polynucleotides (DNA or RNA) encoding the polypeptide ex vivo, and then the cells thus treated are treated with the polypeptide. To the patient. Such methods are well known in the art. For example, cells can be genetically treated by procedures known in the art by using retroviral particles containing RNA encoding the polypeptides of the invention.

Similarly, cells can be engineered in vivo for expression of polypeptides in vivo, for example by procedures known in the art. As is known in the art, producer cells for producing retroviral particles containing RNA encoding the polypeptides of the invention are subjected to a patient to genetically process the cells in vivo and to express the polypeptides in vivo. It can be administered to. Such and other methods of administering a polypeptide of the invention by such a method should be apparent to those skilled in the art from the teachings of the invention. For example, the expression vehicle for manipulating the cell may be other than retroviral particles, for example, which may be used to manipulate the cell in vivo after binding the adenovirus with a suitable transport vehicle.

Retroviruses from which the above-mentioned retroviral plasmid vectors may be derived include, but are not limited to, Moroni murine leukemia virus, spleen necrosis virus, retroviruses such as Raus sarcoma virus, Harvey sarcoma virus, avian leukemia virus, primary ape leukemia Viruses, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and breast tumor virus. In one embodiment, such retroviral plasmid vectors are derived from Moroni murine leukemia virus.

The vector comprises one or more promoters. Suitable vectors that can be used include retroviral LRT; SV40 promoter; And human cytomegalovirus (CMV) promoters described in Miller et al., Biotechniques 7: 980: 990 (1989), or other promoters (eg, histone, pol III and b-actin promoters). However, there are intracellular promoters such as eukaryotic intracellular promoters, which are not limited thereto, but are not limited thereto. Other viral promoters that can be used include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. Selection of suitable promoters will be apparent to those skilled in the art from the teachings contained herein.

Nucleic acid sequences encoding polypeptides of the invention are under the control of suitable promoters. Suitable promoters that can be used include adenovirus promoters such as adenovirus major rate promoters; Heterologous promoters such as cytomegalovirus (CMV) promoters; Respiratory syncytial virus (RSV) promoter; Inducible promoters such as the MMT promoter which is a metallothionein promoter; Heat shock promoter; Albumin promoter; ApoAI promoter; Human globin promoter; Viral thymidine kinase promoters such as the simple herpes thymidine kinase promoter; Retroviral LTRs (including the modified retroviral LTRs described above); b-actin promoter; And human growth hormone promoters. The promoter may be an original promoter that regulates the gene encoding the polypeptide.

Packaging cell lines are transduced using retroviral plasmid vectors to form producer cell lines. Examples of packaging cells that can be transfected include PE501, PA317, y-2, y as described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated by reference in its entirety. -AM, PA12, T19-14X, VT-19-17-H2, yCRE, yCRIP, GP + E-86, GP + envAm12, and DNA cell lines, but are not limited thereto. The vector transduces the packaging cells by any means known in the art. Such means include, but are not limited to, electrophoresis, the use of liposomes, and CaPO ₄ precipitation. In one alternative embodiment, the retroviral plasmid vector can be encapsulated into liposomes or coupled with a lipid and then administered to a host.

Producer cell lines produce infectious retroviral vector particles comprising nucleic acid sequence (s) encoding the polypeptide. The retroviral vector particles are then used to transduce eukaryotic cells in vitro or in vivo. Such transduced eukaryotic cells will express nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic liver cells, embryonic carcinoma cells, and hematopoietic stem cells, liver cells, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

Diagnostic assay

The invention also relates to the use of the VEGF-2 gene as part of a diagnostic assay for detecting a disease associated with or detecting susceptibility to the presence of a mutant in the VEGF-2 nucleic acid sequence.

Individuals involving mutations in the VEGF-2 gene can be detected at the DNA level by various techniques. Diagnostic nucleic acids can be obtained from cells of a patient, such as blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection or can be enzymatically amplified using PCR prior to analysis (Saki et al., Nature 324: 163-166 (1986)). RNA or DNA can also be used for the same purpose. As one example, PCR primers complementary to nucleic acids encoding VEGF-2 can be used to identify and analyze VEGF-2 mutants. For example, deletions and inserts can be detected by changing the size of the amplified product compared to normal genotypes. Point mutations can be identified by hybridizing amplified DNA to radiolabeled VEGF-2 RNA or radiolabeled VEGF-2 antisense DNA sequence. Preferably, the matched sequence can be distinguished from mismatched duplexes by RNaseA digestion or melting point differences.

Genetic testing based on DNA sequence differences can be accomplished by detecting changes in electrophoretic motility of DNA fragments in gels with or without denaturants. Small sequence deletions and inserts can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on modified formamide gradient gels where the motility of the different DNA fragments is delayed in the gel at different locations depending on their specific melting or partial melting points. See, Myers et al., Science 230. : 1242 (1985).

Sequence changes at specific positions can also be indicated by nuclease protection assays such as RNase and S1 protection or chemical cleavage methods (Cotton et al., PNAS, USA 85: 4397-4401 (1985)).

Thus, detection of specific DNA sequences can be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (eg, restriction fragment length polymorphism (RFLP)) and Southern blotting of genomic DNA. Can be.

In addition to the more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The present invention also relates to diagnostic assays for detecting altered levels of VEGF-2 protein in various tissues, which may be due to overexpression of proteins as compared to normal control tissue samples, to determine whether or not they are susceptible to such diseases. Whether or not, for example, abnormal cell differentiation can be detected. Assays used to detect levels of VEGF-2 protein in samples derived from the host are well known to those skilled in the art and include radioimmunoassays, competitive binding assays, western blot analysis, ELISA assays, and "sandwich". ) "Assay. ELISA assays (Coligan, et al., Current Protocols in Immunology, 1 (2), Chapter 6, (1991)) first produce antibodies specific for the VEGF-2 antigen, preferably monoclonal antibodies. It includes. Reporter antibodies are also prepared against these monoclonal antibodies. To such reporter antibodies are attached radioactive, fluorescent, or detectable reagents such as horseradish peroxidase enzymes in this example. The sample is removed from the host and incubated on a solid support (polystyrene dish) that binds to the protein in the sample. The free protein binding site on the dish is then covered by incubating with a nonspecific protein such as bovine serum albumin. Monoclonal antibodies are then incubated in these dishes for the time that these monoclonal antibodies are attached to all VEGF-2 proteins attached to the polystyrene dishes. All unbound monoclonal antibodies are removed by washing with buffer. The reporter antibody is bound to the monoclonal antibody bound to VEGF-2 by placing the reporter antibody bound to horseradish peroxidase in a dish. The unattached reporter antibody is then washed away. The amount developed within a given time by adding the peroxidase substrate to the dish is then a measure of the amount of VEGF-2 protein present in the given patient sample volume as compared to the standard curve.

Competitive assays can be used, wherein an antibody specific for VEGF-2 is attached to a solid support. The polypeptide of the present invention is labeled, for example, by radioactivity, a sample derived from the host is passed over the solid support, and the amount of label detected by, for example, liquid scintillation chromatography, of the VEGF-2 in the sample. It can be correlated with the amount.

The "sandwich" assay is similar to the ELISA assay. In a “sandwich” assay, VEGF-2 is passed over a solid support to bind to an antibody attached to the solid support. The second antibody is then bound to VEGF-2. The third antibody labeled and specific for the second antibody can then be passed over the solid support to bind to the second antibody and then quantified.

Chromosome Identification

The sequences of the invention are also useful for chromosomal identification. The sequence can be specifically targeted to and hybridized to a specific location on an individual human chromosome. Furthermore, there is currently a need to identify specific sites on chromosomes. Very few chromosome labeling reagents are currently available based on actual sequence data (repeat polymorphism) for marking chromosomal positions. Indicating the gene location of the DNA relative to the chromosome according to the invention (mapping) is an important first step in correlating these sequences with the genes associated with the disease.

In brief, sequences can be mapped to chromosomes by making PCR primers (preferably 15-25 bp) from cDNA. Computer analysis of the cDNA is used to quickly select one or more exons in the genomic DNA to complicate the amplification process. The primers are then used for PCR screening of celiac cell hybrids containing individual human chromosomes. Only hybrids containing human genes corresponding to these primers will produce amplified fragments.

PCR mapping of celiac cell hybrids is a rapid method for distributing specific DNA to specific chromosomes. Using the same oligonucleotide primers in the present invention, sub-localization can be achieved in a similar manner using panels of fragments from pools of specific chromosomes or large genomic clones. Other mapping methods that can be similarly used to map to their chromosomes include hybridization in situ, prescreening and hybridization using labeled flow-classified chromosomes to construct chromosomal specific-cDNA libraries. There is a preliminary selection method by.

The cDNA clones can be fluorescently hybridized (FISH) in situ with the mid-term chromosomal diffusion culture to provide the correct chromosome location in one step. This technique can be used with cDNAs as short as 50 or 60 base pairs. For a review of these techniques, reference may be made to Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once the sequence is mapped to the correct chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online via the Johns Hopkins University Welch Medical Library). Next, the relationship between the gene mapped to the same chromosomal region and the disease is identified through a binding analysis (cogenetics of physically adjacent genes).

Next, it is necessary to determine the cDNA or genomic sequence differences between the infected and non-infected. If mutations are observed in some or all infected individuals but not in normal individuals, these mutations are probably the causative agent of the disease.

With conventional physical mapping decomposition and genetic mapping techniques, a cDNA correctly localized to the chromosomal region associated with the disease may be one of 50 to 500 potent causative genes (this is a 1 megabase mapping) Resolution and one gene per 20 kb).

Comparison of infected and non-infected generally involves first looking for structural modifications within the chromosome, such as detectable deletions or translocations that can be detected using PCR based on cDNA sequences or are visible from chromosomal diffusion cultures. Ultimately, complete sequencing of genes from several individuals requires identifying the presence of mutations and distinguishing them from polymorphisms.

Antisense

The invention further relates to the inhibition of VEGF-2 in vivo by using antisense technology. Antisense technology is used to regulate gene expression via antisense DNA or RNA or to regulate gene expression through triple-helix formation. All of these methods are based on binding polynucleotides to DNA or RNA. For example, the 5 'coding portion of a mature polynucleotide sequence encoding a polypeptide of the invention can be used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. By designing DNA oligonucleotides to be complementary to gene regions involved in transcription [triple helix-Lee et al., Nucleic. Acids Research, 6: 3073 (1979); Cooney et al, Science, 241: 456 (1988); And Dervan et al., Science, 251: 1360 (1991), which inhibit the transcription and production of VEGF-2. Antisense RNA oligonucleotides hybridize with mRNA in vivo and block the mRNA molecule from being translated into VEGF-2 [antisense-Okano, J. Neurochem., 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)".

In addition, the aforementioned oligonucleotides are delivered to cells by procedures in the art, such that antisense RNA or DNA can be expressed in vivo to inhibit the production of VEGF-2 in the manner mentioned above.

Thus, antisense constructs for VEGF-2 may inhibit the angiogenic activity of VEGF-2 and may prevent further growth or even inhibit solid tumors, which angiogenesis and neovascularization may produce It's an essential step for growth. These antisense constructs can also be used to treat rheumatoid arthritis, psoriasis, diabetic retinopathy and Kaposi's sarcoma, all characterized by abnormal angiogenesis.

Epitope-retaining portion

In another aspect, the invention provides peptides and polypeptides comprising an epitope-bearing portion of a polypeptide of the invention. Such epitopes are immunogenic or antigenic epitopes of the polypeptides of the invention. An “immunogenic epitope” is defined as a specific portion of a protein that elicits an antibody response in vivo when the complete polypeptide or fragment thereof of the invention is an immunogen. On the other hand, certain regions of the polypeptide to which an antibody can be bound are defined as "antigen determinants" or "antigenic epitopes." The number of immunogenic epitopes in vivo in proteins is generally less than the number of antigenic epitopes. See Geysen, et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983). However, antibodies can be made against any antigenic epitope by using methods such as phage display, regardless of whether it is an immunogenic epitope. See Petersen G. et al. (1995) Mol. Gen. Genet. 249: 425-431. Thus, the present invention includes both immunogenic and antigenic epitopes.

In particular, it is emphasized that immunogenic epitopes include predetermined critical amino acid residues determined by James-Wolf analysis. Thus, additional flanking residues for the N-terminus, C-terminus, or both N and C-terminus can be added to these sequences to produce the epitope-bearing polypeptides of the invention. Thus, immunogenic epitopes may comprise additional N-terminal or C-terminal amino acid residues. The additional flanking amino acid residues may be contiguous flanking N-terminal and / or C-terminal sequences, heterologous polypeptide sequences from a polypeptide of the invention, or may comprise contiguous flanking sequences and heterologous polypeptide sequences from a polypeptide of the invention. It may include.

Polypeptides of the invention comprising an immunogenic or antigenic epitope are seven or more amino acid residues in length. “Abnormal” means that a polypeptide of the invention comprising an immunogenic or antigenic epitope may be seven amino acid residues in length, or any integer between seven amino acids and the number of amino acid residues of the full length polypeptide of the invention. It means that there is. Preferred polypeptides comprising immunogenic or antigenic epitopes are 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues. However, any respective integer between 7 and the number of amino acid residues of the full length polypeptide is included in the present invention.

Immunogenic and antigenic epitope-bearing fragments can be specified by the number of contiguous amino acid residues as mentioned above, or by the N-terminal and C-terminal positions of these fragments on the amino acids of SEQ ID NO: 2. For example, any combination of N-terminal and C-terminal positions where fragments of at least 7 or 15 or more contiguous amino acid residue lengths can be occupied on the amino acid sequence of SEQ ID NO: 2 is included in the invention. In other words, “adjacent amino acid residues of at least seven lengths” means any integer between seven amino acid residues in length, or the number of seven amino acids and amino acid residues of a full length polypeptide of the present invention. Specifically stated, any integer between 7 and the number of amino acid residues of the full length polypeptide is included in the present invention.

Immunogenic and antigenic epitope bearing polypeptides of the invention are useful, for example, in preparing antibodies that specifically bind polypeptides of the invention and in immunoassays to detect polypeptides of the invention. Such antibodies are useful, for example, for affinity purification of the present polypeptides. Antibodies can also be routinely used in various qualitative or quantitative immunoassays, particularly for polypeptides of the invention, using methods known in the art. See Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press; 2nd Ed. 1988).

The epitope bearing polypeptides of the present invention can be prepared by conventional polypeptide production methods, including synthetic and recombinant methods known in the art. For example, epitope bearing peptides can be synthesized using known chemical synthesis methods. For example, Houghten describes a simple method of synthesizing multiple peptides, for example, 10-20 mg of 248 individual peptides and 13 separate residue peptides representing single amino acid variants of fragments of HA1 polypeptides. All of them were prepared and characterized in less than 4 weeks (by ELISA type binding studies). See Houghten, RA Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985). A method for "simultaneous multiple peptide synthesis (SMPS)" is further described in US Pat. No. 4,631,211 (Houghten et al .; 1986). In this process, the individual resins for solid phase synthesis of the various peptides are contained in separate solvent-permeable packets, making optimal use of many of the same repeating steps involved in the solid phase process. In a completely manual process, more than 500 to 1000 syntheses can be performed simultaneously. Houghten et al. (1985) Proc. Natl. Acad. Sci. 82: 5131-5135 at 5134.

Epitope-bearing polypeptides of the invention are used to induce antibodies according to methods well known in the art, including, but not limited to, in vivo immunity, in vitro immunity and phage display methods. Sutcliffe et al., Supra; Wilson et al., Supra; and Bittle, F.J. et al., J. Gen. Virol. 66: 2347-2354 (1985). If in vivo immunity is used, animals can be immunized with free peptides, but anti-peptide antibody titers can be enhanced by coupling the peptides to large molecule carriers such as keyhole limpet hemacyanine (KLH) or tetanus toxin. have. For example, cysteine containing peptides can be coupled to the carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides are more common such as glutaraldehyde. Coupling agents may be used to couple to the carrier. Animals such as rabbits, rats, and mice use free peptides or carrier-coupled carriers, for example, by intraperitoneal and / or intradermal injection of an emulsion containing about 100 mg peptide or carrier peptide and Freund's auxiliary solution. Immunize For example, several booster injections may be needed, eg, at about two week intervals, to provide useful titers of anti-peptide antibodies that can be detected by ELISA assays using free peptides adsorbed on solid surfaces. have. The titer of anti-peptide antibodies in serum from immunized animals is increased by selection of anti-peptide antibodies, for example by adsorbing to peptides on solid supports and eluting the selected antibodies according to methods well known in the art. You can.

Those skilled in the art recognize that polypeptides of the invention, including immunogenic or antigenic epitopes, can be fused with heterologous polypeptide sequences and are also discussed above. For example, a polypeptide of the invention may be fused with a constant region of an immunoglobulin (IgA, IgE, IgG, IgM), or a portion thereof (CH1, CH2, CH3, a combination thereof comprising both complete regions, and portions thereof). To generate a chimeric polypeptide. These fusion proteins promote purification and increase half-life in vivo. This has been shown for chimeric proteins consisting of various regions of the first two regions of human CD4-polypeptide and the constant regions of the heavy or light chains of mammalian immunoglobulins (see EP A 394,827; Traunecker et al., Nature 331: 84-86 (1988). Fusion proteins with disulfide-linked chimeric structures (due to the IgG moiety) may also be more efficient at binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. Fountoulakis et al., J. Biochem . 270: 3958-3964 (1995). The nucleic acid encoding the epitope may also be recombined with the gene of interest as an epitope tag to aid in the detection and purification of the expressed polypeptide.

Antibodies

The invention further relates to antibodies and T-cell antigen receptors (TCRs) that specifically bind to polypeptides of the invention. Antibodies of the invention include IgG (including IgG1, IgG2, IgG3 and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM and IgY. As used herein, the term “antibody” (Ab) means that the complete molecule, the complete molecule of the single chain and the antigen-binding fragment thereof are included. More preferably, the antibody is a human antigen binding antibody fragment of the invention, which includes Fab, Fab 'and F (ab') ₂ , Fd, single chain Fvs (scFv), single chain antibody, disulfide-linked Fvs fragments comprising (sdFv) and a V _L or V _H region are included, but are not limited thereto. The antibody may be of any animal origin, including birds and mammals. Preferably, the antibody is of human, rat, rabbit, goth, guinea pig, camel, horse or chicken origin.

Antigen-binding antibody fragments, including single chain antibodies, may comprise the variable region (s) alone or in combination with all or a portion of the following regions: hinge region, CH1, CH2 and CH3 regions. Also included in the present invention are any combinations of variable regions and hinge regions, CH1, CH2 and CH3 regions. The invention further includes chimeric, humanized and human monoclonal and polyclonal antibodies that specifically bind to the polypeptides of the invention. The invention further includes antibodies that are anti-genetic to the antibodies of the invention.

Antibodies of the invention may be monospecific, bispecific, triple specific or more multi specific. Multispecific antibodies may be specific for different epitopes of the polypeptides of the invention or may be specific for both the polypeptides of the invention as well as for heterologous compositions such as heterologous polypeptides or solid phase materials. WO 93 / 17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991) J. Immunol. 147: 60-69; U.S. Patents 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S.A. et al. (1992) J. Immunol. 148: 1547-1553.

Antibodies of the invention may be described or embodied in terms of epitope (s) or portion (s) of the polypeptides of the invention that are recognized by or specifically bound by such antibodies. Epitope (s) or polypeptide portion (s) can be specified by N-terminal and C-terminal positions, the size of contiguous amino acid residues, or as opened in the tables and figures, as described herein. Antibodies that specifically bind to any epitope or polypeptide of the invention may also be excluded. Thus, the invention includes antibodies that specifically bind to and allow exclusion of the polypeptides of the invention.

Antibodies of the invention may also be described or embodied in terms of their cross-reactivity. Antibodies that do not bind any other homologs, orthologs or homologues of the polypeptides of the invention are also included. Less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55% and less than 50% identity with the polypeptides of the present invention (known in the art) Also included in the present invention are antibodies that do not bind to a polypeptide that is identifiable as defined using the methods described above. In addition, the present invention includes antibodies that bind only to polypeptides encoded by polynucleotides that hybridize with the polynucleotides of the present invention under stringent hybridization conditions (as described above). Antibodies of the invention may also be described or specified in terms of their binding affinity. Preferred binding affinity is 5X10 ^-6 M, 10 ^-6 M, 5X10 ^-7 M, 10 ^-7 M, 5X10 ^-8 M, 10 ^-8 M, 5X10 ^-9 M, 10 ^-9 M, 5X10 ^-10 M, 10 ^-10 M, 5X10 ^-11 M, 10 ^-11 M, 5X10 ^-12 M, 10 ^-12 M, 5X10 ^-13 M, 10 ^-13 M, 5X10 ^-14 M, 10 ^-14 M, 5X10 ^-15 M and 10 ^{And those} having a dissociation constant or Kd of less than ^-15 M.

Antibodies of the invention have uses including, but not limited to, methods known in the art for purifying, detecting, and targeting polypeptides of the invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has use in immunoassays to qualitatively and quantitatively determine the degree of polypeptides of the invention in biological samples. See Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor. Laboratory Press, 2nd ed. 1988); Incorporated herein by reference.

Antibodies of the invention can be used alone or in combination with other compositions. The antibody may further be recombinantly fused with the heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugation) to the polypeptide or other composition. For example, antibodies of the invention can be recombinantly fused or conjugated with molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs or toxins. See WO 92/08495; WO 91/14438; WO 89/12624; US Patent No. 5,314,995; And EP 0 396 387]. Antibodies of the invention can be prepared by any suitable method known in the art. For example, the polypeptide of the invention or antigenic fragments thereof can be administered to an animal to induce the production of serum containing polyclonal antibodies. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas and recombinant techniques. See Harlow et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory. Press, 2nd ed. 1988); Hammerling, et al., In: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, NY, 1981); Incorporated herein by reference. Fab and F (ab ') 2 fragments are produced by proteolytic cleavage using enzymes such as papain (to generate Fab fragments) or pepsin (to generate F (ab') ₂ fragments).

Alternatively, the antibodies of the invention can be prepared via synthetic chemistry, either by the application of recombinant DNA techniques or using methods known in the art. For example, the antibodies of the present invention can be prepared using various phage display methods known in the art. In phage display methods, functional antibody regions are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. By directly selecting antigens, typically antigens bound or captured to solid surfaces or beads, phages with the desired binding properties are selected from repertoires or combinatorial antibody libraries (eg human or murine). Phage used in this method are typically filamentous phage comprising fd and M13 with Fab, Fv or disulfide stabilized Fv antibody regions recombinantly fused to phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare the antibodies of the invention include those described in Brinkman U. et al. (1995) J. Immunol. Methods 182: 41-50; Ames, R.S. et al. (1995) J. Immunol. Methods 184: 177-186; Kettleborough, C. A. et al. (1994) Eur. J. Immunol. 24: 952-958; Persic, L. et al. (1997) Gene 187 9-18; Burton, D. R. et al. (1994) Advances in Immunology 57: 191-280; PCT / GB 91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; And 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,585,658 And 5,733,743; All of which are incorporated herein by reference in their entirety.

As described in the references above, after phage selection, the antibody coding region from the phage is isolated and used to generate complete antibodies, including human antibodies or other desired antibody binding fragments, mammalian cells, insect cells, plant cells It can be expressed in any host, including yeast and bacteria. For example, techniques for recombinantly preparing Fab, Fab 'and F (ab') 2 fragments may also be used using methods known in the art as described in the following references. WO 92 / 22324; Mullinax, R.L. et al. (1992) BioTechniques 12 (6): 864-869; and Sawai, H. et al. (1995) AJRI 34: 26-34; and Better, M. et al. (1988) Science 240: 1041-1043; Incorporated herein by reference.

Examples of techniques that can be used to prepare single chain Fvs and antibodies include those described in the following references: US Pat. Nos. 4,964,778 and 5,258,498; Huston et al. (1991) Methods in Enzymology 203: 46-88; Shu, L. et al. (1993) PNAS 90: 7995-7999; and Skerra, A. et al. (1988) Science 240: 1038-1040. For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be desirable to use chimeric, humanized or human antibodies. Methods of making chimeric genes are known in the art. See Morrison, Science 229: 1202 (1985); Oi, et al., BioTechniques 4: 214 (1986); Gillies, S.D. et al. (1989) J. Immunol. Methods 125: 191-202; and US Pat. No. 4,816,567]. Antibodies are CDR-grafted [EP 0 239 400; WO 91/09967; US Pat. Nos. 5,530,101 and 5,585,089], appended or resurfaced [EP 0592 106; EP 0 519 596; Padlan E. A., (1991) Molecular Immunology 28 (4/5): 489-498; Studnicka G.M. et al. (1994) Protein Engineering 7 (6): 805-814; Roguska M.A. et al. (1994) PNAS 91: 969-973, chain shuffling (US Pat. No. 5,565,332), and may be adapted to human adaptation. Human antibodies can be made by a variety of methods known in the art, including the phage display ice methods mentioned above (see, eg, US Pat. Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318 and WO 98/46645; Incorporated herein by reference.

Further included in the present invention are antibodies that have been recombinantly fused to or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide of the invention. Such antibodies may be specific for antigens other than the polypeptides of the invention. For example, an antibody can be used to target a polypeptide of the invention to a particular cell type, either in vitro or in vivo, by fusion or conjugation of the polypeptide of the invention to an antibody specific for a particular cell surface receptor. Antibodies fused or conjugated to the polypeptides of the present invention can be used for in vitro immunoassay and purification methods using methods known in the art. See Harbor et al. See above and WO 93/21232; EP 0 439 095; Naramura, M. et al. (1994) Immunol. Lett. 39: 91-99; US Patent No. 5,474,981; Gillies, S.O. et al. (1992) PNAS 89: 1428-1432; Fell, H.P. et al. (1991) J. Immunol. 146: 2446-2452; Incorporated herein by reference.

The present invention further includes compositions comprising polypeptides of the invention fused or conjugated to antibody regions other than variable regions. For example, a polypeptide of the invention can be fused or conjugated to an antibody Fc region or portion thereof. Antibody moieties fused to a polypeptide of the invention may comprise a hinge region, a CH1 region, a CH2 region and a CH3 region, or any combination of complete regions or portions thereof. Polypeptides of the invention can be fused or conjugated to such antibody portions to increase the in vivo half-life of such polypeptides or for use in immunoassays using methods known in the art. Polypeptides of the invention can also be fused or conjugated to the antibody moiety to form multimers. For example, an Fc moiety fused to a polypeptide of the invention can form a dimer through disulfide bonds between the Fc moieties. Higher order multimeric forms can be made by fusing the polypeptides of the invention to portions of IgA and IgM. Methods of fusion or conjugation of polypeptides of the invention to antibody portions are known in the art. See, eg, US Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al. (1991) PNAS 88: 10535-10539; Zheng, X.X. et al. (1995) J. Immunol. 154: 5590-5600; and Vil. H. et al. (1992) PNAS 89: 11337-11341; Incorporated herein by reference.

The invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the invention. For example, the present invention includes antibodies that partially or completely disrupt receptor / ligand interactions with polypeptides of the invention. Both receptor-specific and ligand-specific antibodies are included. Receptor specific antibodies that do not inhibit ligand binding but inhibit receptor activation are included. Receptor activation (signaling) can be determined by techniques described herein or by techniques known in the art. Also included are receptor-specific antibodies that inhibit both ligand binding and receptor activation. Similarly, neutralizing antibodies that bind to the ligand to inhibit binding of the ligand to the receptor, as well as antibodies that inhibit receptor activation by binding to the ligand but do not inhibit the binding of the ligand to the receptor are included. In addition, antibodies that activate the receptor are included. These antibodies can act as agonists for all or less of the biological activity affected by ligand mediated receptor activation. The antibody can be specified as an agonist or antagonist for biological activity, including the specific activity described herein. Such antibody agonists can be prepared using methods known in the art. See WO 96/40281; US Patent No. 5,811,097; Deng, B. et al. (1998) Blood 92 (6); 1981-1988; Chen, Z. et al. (1998) Cancer Res. 58 (16): 3668-3678; Harrop, J. A. et al. (1998) J. Immunol. 161 (4): 1786-1794; Zhu, Z. et al. (1998) Cancer Res. 58 (15): 3209-3214; Yoon, D.Y. et al. (1998) J. Immunol. 160 (7): 3170-3179; Prat, M. et al. (1998) J. Cell. Sci. 111 (Pt 2): 237-247; Pitard, V. et al. (1997) J. Immunol. Methods 205 (2): 177-190; Liautard, J. et al. (1997) Cytokinde 9 (4): 233-241; Carlson, N.G. et al. (1997) J. Biol. Chem. 272 (17): 11295-11301; Taryman, R. E. et al. (1995) Neuron 14 (4): 755-762; Muller, Y.A. et al. (1998) Structure 6 (9): 1153-1167; Bartunek, P. et al. (1996) Cytokine 8 (1): 14-20; Incorporated herein by reference.

Antibodies can be used in immunoassays to detect the presence of tumors in certain individuals. Enzyme immunoassays can be performed from blood samples of specific individuals. Increased levels of VEGF-2 can be considered to diagnose cancer.

Truncated versions of VEGF-2 may be prepared that do not activate endothelial cell growth and thus can interact with wild type VEGF-2 to form dimers that inactivate endogenous VEGF-2. Alternatively, VEGF-2 in mutant form forms the dimer itself and occupies the ligand binding region of the appropriate tyrosine kinase receptor on the target cell surface, but does not activate cell growth.

Alternatively, an antagonist of the polypeptide of the invention that binds to a receptor that normally binds the polypeptide of the invention can be used. Because these antagonists are closely related proteins, they recognize and bind the receptor sites of natural proteins, but because they are inactive forms of natural proteins, they inhibit the action of VEGF-2 because the receptor sites are occupied. In these ways, the action of VEGF-2 is inhibited and the antagonist / inhibitor is used therapeutically as an anti-tumor drug by occupying a receptor site in a tumor recognized by VEGF-2 or by inactivating VEGF-2 itself. Can be. This antagonist / inhibitor can also be used to prevent bleeding due to increased vascular permeability action of VEGF-2. The antagonist / inhibitor can also be used to treat solid tumor growth, diabetic retinopathy, psoriasis and rheumatoid arthritis.

The antagonist / inhibitor can be used in the composition with a pharmaceutically acceptable carrier as described above.

The invention will be further described with reference to the following examples, but the invention is not limited to these examples. All parts or amounts are by weight unless otherwise indicated.

In order to facilitate understanding of the following examples, specific methods and / or terminologies are often described.

"Plasmids" are written first in lowercase p and / or in uppercase and / or numerals. Starting plasmids in the present invention are commercially available for open purchase under unrestricted conditions or can be constructed according to methods published from commercial plasmids. In addition, plasmids equivalent to those described herein are known in the art and will be apparent to those skilled in the art.

"Digestion" of DNA refers to catalytic cleavage of DNA with restriction enzymes that act only on specific sequences of DNA. Various restriction enzymes used in the present invention are commercially available and their reaction conditions, cofactors and other requirements are used as known to those skilled in the art. For analytical purposes, typically 1 mg of plasmid or DNA is used with about 2 units of enzyme in about 20 Fl of buffer. If DNA fragments are to be isolated for plasmid construction, typically 5-50 mg of DNA is digested into larger volumes using 20-250 units of enzyme. The amount of buffer and substrate suitable for the particular restriction enzyme is specified by the manufacturer. Incubation times of about 1 hour at 37 ° C. are conventionally used but may be varied as directed by the supplier. After digestion, the reaction is electrophoresed directly on a polyacrylamide gel to separate the desired fragments.

Size separation of the cleaved fragments is performed using an 8% polyacrylamide gel described in Goeddel, D. et al., Nucleic Acids Res., 8: 4057 (1980).

"Oligonucleotide" refers to single-chain polydeoxynucleotides or two complementary polydeoxynucleotide chains that can be chemically synthesized. Such synthetic oligonucleotides do not have 5 'phosphate and thus are not linked to another oligonucleotide unless phosphate is added with ATP in the presence of a kinase. Synthetic oligonucleotides will be linked to fragments that are not dephosphorylated.

"Ligation" refers to the process of forming a phosphodiester bond between two double-stranded nucleic acid fragments. See Sambrook. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989), p. 146]. Unless otherwise indicated, ligation can be performed using well known buffers and conditions using approximately 10 equimolar amounts of 10 units of T4 DNA ligase (“ligase”) per 0.5 mg of DNA fragment to be linked.

Unless stated otherwise, transformation is performed as described in Graham, F. and Van der Eb, A., Virology, 52: 456-457 (1973).

Example 1 Expression Patterns of VEGF-2 in Human Tissues and Breast Cancer Cell Lines

Northern blot analysis is performed to examine the expression of VEGF-2 in breast cancer cell lines in human tissues and human tissues. Total intracellular RNA cells are isolated using RNAzol ™ B system (Biotecx Laboratories). 10 mg of total RNA isolated from each characterized breast tissue and cell line are separated on a 1% agarose-formaldehyde gel and blotted onto a nylon filter. Sambrook. et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989). Labeling reactions are performed according to the Stratagen Primer-It Kit using 50ng DNA fragments. The labeled DNA is purified by Select-G-50 column (5 Prime-3 Prime, Inc. (Boulder, CO)). The filter is then hybridized with full length VEGF-2 gene radiolabeled at 1,000,000 cpm / ml in 0.5M NaPO ₄ and 7% SDS overnight at 65 ° C. After washing twice at room temperature with 0.5 × SSC, 0.1% SDS and twice at 60 ° C., the filter is exposed overnight at −70 ° C. using a concentration screen. A message of 1.6Kd was found in two breast cancer cell lines. 5, lane 4 shows an extremely oncogenic cell line that is growth-independent estrogen.

In addition, 10 mg total RNA from 10 human adult tissues are separated on agarose gels and blotted onto nylon filters. The filter was then washed overnight at 65 ° C. with 7% SDS, 0.5M NaPO ₄ , pH 7.2; Hybridize with radiolabeled VEGF-2 probe in 1% BSA. After washing with 0.2 × SSC at 65 ° C., the filter is exposed to film for 24 days at −70 ° C. using a concentrating screen. See FIG. 6.

Example 2: Expression of Truncated Form of VEGF-2 (SEQ ID NO: 4) by In Vitro Transcription and Detoxification

VEGF-2 cDNAs are transcribed and translated in vitro to determine the size of the translatable polypeptide encoded by the truncated forms of VEGF-2 and partial VEGF-2 cDNA. Two inserts of VEGF-2 in the pBluescript SK vector are amplified by PCR using the following three pairs of primers: 1) M13-reverse and forward primers; 2) M13-reverse primer and VEGF primer F4; And 3) M13-reverse primer and VEGF primer F5. The sequence of these primers is as follows.

M13-2 backward primer: 5'-ATGCTTCCGGCTCGTATG-3 '(SEQ ID NO: 9). This sequence is located on top of the 5 'end of the VEGF-2 cDNA insert in the pBluescript vector and is in antisense orientation as cDNA. The T3 promoter sequence is located between the primer and the VEGF-2 cDNA. M13-2 forward primer: 5'-GGGTTTTCCCAGTCACGAC-3 '(SEQ ID NO: 10). This sequence is located at the bottom of the 3 'end of the VEGF-2 cDNA insert in the pBluescript vector and in antisense orientation as the cDNA insert. VEGF Primer F4: 5'-CCACATGGTTCAGGAAAGACA-3 '(SEQ ID NO: 11). This sequence is located in the VEGF-2 cDNA in antisense orientation from bp 1259-1239, which is about 169 bp from the 3 'end of the stop codon and about 266 bp before the last nucleotide of the cDNA.

PCR reactions using all three pairs of primers result in an amplified product with a T3 promoter sequence in front of the cDNA insert. The first and third pairs of primers produce a PCR product encoding the polypeptide of VEGF-2 set forth in SEQ ID NO: 4. The second pair of primers produces a PCR product that omits the 36 amino acid coding sequence at the C-terminus of the VEGF-2 polypeptide.

About 0.5 mg of PCR product from the first pair of primers, about 1 mg of PCR product from the second pair of primers, and about 1 mg of PCR product from the third pair of primers are used for in vitro transcription / detoxification. This in vitro transcription / detoxification reaction is performed at a volume of 25 Fl using a T _N TJ coupled reticulocyte melt system (Promega, CAT # L4950). Specifically, the reactant is 12.5Fl T _N T rabbit reticulocyte melt, 2Fl T _N T reaction buffer, 1Fl T3 polymerase, 1Fl 1 mM amino acid mixture (excluding methionine), 4Fl ³⁵ S-methionine (> 1000 Ci / mmol, 10 mCi / ml), 1 U of 40 U / μl; RNasin ribonuclease inhibitors, containing 0.5 or 1 mg of PCR product. Nuclease-free H ₂ O is added to bring the volume to 25 Fl. The reaction is incubated at 30 ° C. for 2 hours. Five microliters of reaction product are analyzed on a 4-20% gradient SDS-PAGE gel. After fixation in 25% isopropanol and 10% acetic acid, the gel is dried and exposed to X-ray film overnight at 70 ° C.

As shown in FIG. 7, PCR products containing excised VEGF-2 cDNA (ie as shown in SEQ ID NO: 3) and cDNA omitted 266 bp in the 3 'untranslated region (3'-UTR) are of the same length Produces a product, the molecular weight of which is estimated to be 38-40 dk (lanes 1 and 3). The cDNA omitting the 3′UTR and the omission sequence encoding the C-terminal 36 amino acids are translated into polypeptides with molecular weights estimated to be 36 to 38 kd (lane 2).

Example 3: Cloning and Expression of VEGF-2 Using Baculovirus Expression System

DNA sequences encoding VEGF-2 protein without 46 amino acids at the N-terminus (ATCC Accession No. 97149) are amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene.

The 5 'primer has the sequence 5'-TGT AAT ACG ACT CAC TAT AGG GAT CCC GCC ATG GAG GCC ACG GCT TAT GC (SEQ ID NO: 12) and is complementary to the BamH restriction enzyme site (shown in bold) and the 5' sequence of VEGF-2. It contains 17 nucleotide sequences (nt. 150-166).

The 3 'primer has the sequence GATC TCT AGA TTA GCT CAT TTG TGG TCT (SEQ ID NO: 13) and comprises the cleavage site for restriction endonuclease XbaI, and the 15' sequence before the stop codon and the stop codon, It contains 18 nucleotides complementary to.

Amplified sequences are separated from 1% agarose gels using a commercial kit (trade name: “Geneclean” from BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with endonuclease BamHI and XbaI and purified again on 1% agarose gel. This fragment is linked to the pAcGP67A baculovirus transfer vector (Pharmingen) at the BamHI and XbaI sites. Through this linkage, VEGF-2 cDNA is cloned in the same frame as the signal sequence of the baculovirus gp67 gene and placed at the 3 'end of the signal sequence in the vector. It is named pAcGP67A-VEGF-2.

In order to clone VEGF-2 with the signal sequence of the gp67 gene into the pRG1 vector for expression, VEGF-2 and several top sequences with this signal sequence were located at the Xho restriction endonuclease site located at the top of the VEGF-2 cDNA. And from the pAcGP67A-VEGF-2 plasmid at the XbaI restriction endonuclease site by Xho and XbaI restriction enzymes. This fragment is separated from the rest of the vectors on a 1% agarose gel and purified using the "Geneclean" kit. Call it F2.

The PRG1 vector (a variant of the pVL941 vector) is used for the expression of the VEGF-2 protein using the baculovirus expression system. Summers, M.D. and Smith, G.E. 1987, "A manual of methods for baculovirus vectors and insect cell culture procedures", Texas Agricultural Experimental Station Bulletin No. 1555]. This expression vector contains a potent polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by recognition sites for restriction endonucleases BamHI, SmaI, XbaI, BglII and Asp718. do. The site for restriction endonuclease XhoI is located on top of the BamHI site. The sequence between XhoI and BamHI is identical to that in the PAcGp67A (Static On Tape) vector. The polyadenylation site of monkey virus (SV) 40 is used for efficient polyadenylation. For easy selection of recombinant viruses, E. coli. Beta-galactosidase from E. coli is inserted following the polyhedrin promoter in the same direction as the polyadenylation signal of the polyhedrin gene. Polyhedrin sequences are flanked by viral sequences for cell-mediated homologous recombination of cotransfected wild type viral DNA. Many other baculovirus vectors such as pAc373, pVL941 and pAcIM1 can be used in place of pRG1. See Luckow, V.A. and Summers, M.D., Virology, 170: 31-39 (1989).

The plasmid is digested with restriction enzymes XboI and XbaI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. This DNA is isolated from 1% agarose gel using a commercial kit (trade name: "Geneclean", source: BIO 101 Inc., La Jolla, Ca.).

Fragment F2 and dephosphorylated plasmid V2 are linked using T4 DNA ligase. Then, this. E. coli HB101 cells are transformed and the bacteria containing the plasmid (pBc gp67-VEGF-2) carrying the VEGF-2 gene are identified using the enzymes BamHI and XbaI. The sequence of such cloned fragments is confirmed by DNA sequencing.

5 mg of the plasmid pBac gp67-VEGF-2 was lipofected by Felgner et al., Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)], commercially available linearized baculovirus (trade name: "BaculoGoldTM baculovirus DNA", source: Pharmingen, San Diego, CA.) at 1.0 mg. Cotransfection.

1 mg of baculogold virus DNA and 5 mg of plasmid pBac gp67-VEGF-2 are mixed in sterile wells of microtiter plates containing 50 ml of serum-free Grace medium (Life Technologies Inc., Gaithersburg, MD). Then 10 ml of lipofectin and 90 ml of Grace's medium are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture is then added dropwise with 1 ml of serum free medium to Sf9 insect cells (ATCC CRL 1711) inoculated in a 35 mm tissue culture plate. The plate is shaken back and forth to mix the newly added solution. The plates are then incubated at 27 ° C. for 5 hours. After 5 hours, the transfection solution is removed from the plate and 1 ml of Grace insect medium supplemented with 10% fetal calf serum is added. The plate is placed in an incubator and incubated at 27 ° C. for 4 days.

After 4 days the supernatant is collected and a plaque assay is performed similar to that described by Summers and Smith. As a variant, an agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used that facilitates the separation of blue stained plaques. (Detailed description of “Plaque Assay” may be found in User Guides on pages 9-10 of Insect Cell Culture and Baculovirology distributed by the supplier (Life Technologies Inc., Gaithersburg).

Four days after a series of dilutions are performed, the virus is added to the cells and the blue stained plaques are picked out with an Eppendorf pipette tip. The agar containing the recombinant virus is then resuspended in an Eppendorf tube containing 200 ml of Grace's medium. The agar is removed by short centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells inoculated in 35 mm dishes. After 4 days the supernatant of the culture dish is collected and stored at 4 ° C.

Sf9 cells are grown in Grace medium supplemented with 10% heat-inactivated FBS. The cells are infected with recombinant baculovirus V-gp67-VEGF-2 at multiplicity of infection (MOI) 1. After 6 hours the medium is removed and replaced with SF900 II medium without methionine and cysteine [Life Technologies Inc., Gaithersburg]. After 42 hours ^{35 m} -methionine 5 mCi and ³⁵ S-cysteine 5 mCi (Source: Amersham) are added. The cells are further incubated for 16 hours before harvesting by centrifugation and the labeled proteins are visualized by SDS-PAGE and autoradiography.

Proteins from the media and cytoplasm of Sf9 cells are analyzed by SDS-PAGE under non-reducing and reducing conditions (see FIGS. 8A and 8B, respectively). The medium is dialyzed against 50 mM MES, pH 5.8. After dialysis a precipitate is obtained which is resuspended in 100 mM Na citrate, pH 5.0. The precipitate thus resuspended is analyzed again by SDS-PAGE and stained with Coomassie Brilliant Blue (see FIG. 9).

The media supernatant is also diluted to 1:10 in 50 mM MES, pH 5.8 and applied to a SP-650M column (1.0 x 6.6 cm, Toyopearl) at a flow rate of 1 ml / min. Proteins are eluted in step gradients at 200, 300 and 500 mM NaCl. Elution at 500 mM gives VEGF-2. This eluate is analyzed by SDS-PAGE in the presence or absence of b-mercaptoethanol as a reducing agent and stained with Coomassie Brilliant Blue (see FIG. 10).

Example 4: Expression of Recombinant VEGF-2 in COS Cells

Expression of the plasmid VEGF-2-HA was determined by 1) the origin of SV40 replication, 2) the ampicillin resistance gene, and 3) E. E. coli replication origin, 4) CMV promoter followed by vector pcDNAI / Amp (Invitrogen) containing polylinker region, SV40 intron and polyadenylation site. The DNA fragment encoding the complete VEGF-2 precursor and the HA tag fused in frame at its 3 'end are cloned into the polylinker region of the vector. Thus, recombinant protein expression is directed under the CMV promoter. HA tags correspond to epitopes derived from influenza hemagglutinin proteins as described above (Wilson et al., Cell 37: 767 (1984)). Fusion of the HA tag to the target protein facilitates detection of the recombinant protein using an antibody that recognizes an HA epitope.

Plasmid construction methods are described below:

A DNA sequence encoding VEGF-2 (ATCC No. 97149) is constructed by PCR using the following two primers: 5 'primer (CGC GGA TCC ATG ACT GTA CTC TAC CCA) (SEQ ID NO: 14) is the BamHI site Then contains 18 nucleotides of the VEGF-2 coding sequence starting from the start codon; The 3 'sequence (CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAG GCT CAT TTG TGG TCT 3') (SEQ ID NO: 15) is the last 15 sequences of XbaI site, HA tag, XhoI site and VEGF-2 coding sequence. It contains sequences complementary to nucleotides (does not contain stop codons). Thus, this PCR product contains a BamHI site, coding sequence followed by an XhoI restriction endonuclease site and an HA tag fused in frame, a translational stop codon following this HA tag, and an XbaI site. PCR amplified DNA fragments and vector pcDNAI / Amp are digested and linked with BamHI and XbaI restriction enzymes. This coupling mixture. The E. coli strain SURE (Source: Stratagene Cloning System, 11099 North Torrey Pines Road, La Jolla, CA 92037) is transformed, the transformed cultures are plated on ampicillin medium plates and resistant colonies are selected. Plasmid DNA is isolated from the transformants and examined by restriction analysis to determine the presence of the correct fragments. To express recombinant VEGF-2, COS cells are transfected with expression vectors by the DEAE-DEXTRAN method. J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press , (1989). Expression of the VEGF-2-HA protein is detected by radiolabeling and immunoprecipitation (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Laboratory Press, (1988)). Two days after transfection, cells are labeled with ³⁵ S-cysteine for 8 hours. The culture medium was then collected and cells were washed (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.1% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37: 767 (1984)). Both cell melt and culture medium are precipitated with HA specific monoclonal antibodies. Precipitated protein is analyzed on a 15% SDS-PAGE gel.

Example 5 Effect of Partially Purified VEGF-2 Protein on Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) were treated with M199 medium containing 4% fetal bovine serum (FBS), 16 units / ml heparin, and 50 units / ml endothelial cell growth supplement (ECGS, Biotechnique, Inc.). Inoculate at a 5 × 10 ⁴ cells / 35 mm dish density. On day 2, the medium is replaced with M199 containing 10% FBA, 8 units / ml heparin. The VEGF-2 protein of SEQ ID NO: 2, with the initial 45 amino acid residues omitted, (VEGF) and basic FGF (bFGF) are added at the indicated concentrations. On days 4 and 6, the medium is replaced. On day 8, cells are determined by coulter counters (see FIG. 12).

Example 6: Effect of Purified VEGF-2 Protein on Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) were treated with M199 medium containing 4% fetal bovine serum (FBS), 16 units / ml heparin, and 50 units / ml endothelial cell growth supplement (ECGS, Biotechnique, Inc.). At 2 to 5 x 10 ⁴ cells / 35 mm dish density. On day 2, the medium is replaced with M199 containing 10% FBA, 8 units / ml heparin. At this time, the purified VEGF-2 protein of SEQ ID NO: 2 having the initial 45 amino acid residues omitted is added to the medium. On days 4 and 6, the medium is replaced with fresh medium and supplement. On day 8, cells are determined by coulter counters (see FIG. 13).

Example 7: Expression via Gene Therapy

Fibroblasts are obtained from a skin biopsy subject. The resulting tissue is placed in tissue-culture medium and separated into small pieces. A small piece of tissue is placed on the wet surface of the tissue culture flask and approximately 10 pieces are placed in each flask. The flask is rotated up and down, tightly sealed and left overnight at room temperature. After 24 hours at room temperature, the flask is inverted and the tissue pieces are fixed at the bottom of the flask and fresh medium (eg, Hans F12 medium supplemented with 10% PBS, penicillin and streptomycin) is added. This culture is incubated at 37 ° C. for approximately 1 week. At this time, fresh medium is added and changed continuously every few days. After two more weeks of incubation, fibroblast monolayers appear. This monolayer is trypsinized and weighed into a larger flask.

PMV-7 flanked by long terminal repeats of Moroni murine sarcoma virus, Kirschmeier, P.T. et al, DNA, 7: 219-25 (1988)] are digested with Eco RI and HindIII and subsequently treated with calf intestinal phosphatase. Linear vectors are fractionated on agarose gels and purified using glass beads.

CDNA encoding the polypeptides of the invention are amplified using PCR primers corresponding to the 5 'and 3' terminal sequences, respectively. The 5 'primers and 3' primers containing the EcoRI site further comprise a HindIII site. Equal amounts of Moroni murine sarcoma virus linear extension and amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for linking the two fragments. This linkage mixture is used to transform bacterial HB101 and then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest with the appropriate insertion.

Ampotropic pA317 or GP + am12 packaging cells are grown in tissue culture to confluence density in Dulbeckco modified Eagles medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. . The MSV vector containing the gene is then added to the medium and packaging cells are transduced with the vector. The packaging cells then produce infectious viral particles containing the gene (these packaging cells are referred to as producer cells).

Fresh medium is added to the transduced producer cells, and the medium is subsequently harvested from 10 cm plates of confluent producer cells. The spent medium containing infectious virus particles is filtered through a Millipore filter to remove detached producer cells. This medium is then used to infect fibroblast cells. The medium is removed from the sub-confluent plate of fibroblasts and quickly replaced with medium from producer cells. Remove this medium and replace with fresh medium. If the titer of the virus is high, practically all fibroblasts will be infected and no screening is required. If this titer is very low, it may be necessary to use retroviral vectors with selectable markers such as neo or his.

Genetically engineered fibroblasts are then injected alone into the host, or after growth with confluence on cytodex 3 microcarrier beads. These fibroblasts then produce protein products.

Example 8: Expression of VEGF-2 mRNA in Human Fetus and Adult Tissue

Experimental design

Northern blot analysis is performed to examine the expression level of VEGF-2 mRNA in human fetus and adult tissue. CDNA probes containing the entire nucleotide sequence of the VEGF-2 protein are labeled with ³² P using the rediprime ^™ DNA labeling system (Amersham Life Science) according to the manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN-100 ^* column (Clontech Laboratories, Inc.) according to manufacturer's protocol number PT1200-1. This purified and labeled probe is used to test various human tissues for VEGF-2 mRNA.

Multiple tissue northern (MTN) blots containing various human tissues (fetal kidney, fetal lung, fetal liver, brain, kidney, lung, liver, spleen, thymus, bone marrow, testes, placenta and skeletal muscle) were obtained from Clontech Test with a labeled probe using ExpressHyb ^™ Hybridization Solution (Clontech) according to protocol number PT1190-1. After performing the hybridization and washing, the blot is exposed to the film overnight at −70 ° C. with a concentration screen and the film is developed according to standard procedures.

result

Expression of VEGF-2 mRNA is disrupted in vascular smooth muscle and some highly vascularized tissues. VEGF-2 is expressed at significantly higher levels in tissues associated with hematopoietic or angiogenic activity, namely fetal kidney, fetal lung, bone marrow, placenta, spleen and lung tissue. Expression levels of VEGF-2 are low in adult kidney, fetal liver, adult liver and testes; It is rarely detectable in fetal and adult brains (see FIG. 14).

In primary cultured cells, expression of VEGF-2 mRNA is abundant in various smooth muscle cells and intradermal fibroblasts, but lower in human umbilical vein endothelial cells (see FIG. 15). This mRNA distribution pattern is very similar to that of VEGF.

Example 9: Construction of Amino and Carboxy Terminal Deletion Variants

To identify and analyze biologically active VEGF-2 polypeptides, a panel of deletion mutants of VEGF-2 is constructed using the expression vector pHE4a.

1. Construction of VEGF-2 T103-L215 at pHE4

Amplified Directed Polymerase Chain Reaction and E. To allow sub-cloning of VEGF-2 T103-L215 (amino acids 103-215 in FIG. 1 or SEQ ID NO: 18) into E. coli protein expression vector pHE4, complementary to the desired region of VEGF-2, having the following base sequence: Synthesize two oligonucleotide primers:

5 'primer (coding sequences of NdeI / START and 18nt):

5'-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3 '(SEQ ID NO: 19)

3 ′ primer (coding sequences of Asp718, STOP, and 15nt):

5'-GCA GCA GGT ACC TCA CAG TTT AGA CAT GCA-3 '(SEQ ID NO: 20).

The above-mentioned 5 'primer (SEQ ID NO: 19) contains an NdeI restriction site and the above-mentioned 3' primer (SEQ ID NO: 20) contains an Asp718 restriction site. The 5 'primer (SEQ ID NO: 19) is also known as E. coli. The 3 'primer (SEQ ID NO: 20) contains the ATG sequence adjacent to the VEGF-2 coding region and within the same frame, allowing translation of the cloned fragment in E. coli. It contains one stop codon (preferably used in E. coli) in the same frame adjacent to the VEGF-2 coding region, which ensures correct translation termination in E. coli.

The polymerase chain reaction is carried out using standard conditions well known to those skilled in the art and using the nucleotide sequence of the mature VEGF-2 (amino acids 24 to 419 in SEQ ID NO: 18) as a template, as constructed in Example 3. . The resulting amplicon is subjected to restriction digestion with NedI and Asp718 and subcloned into the NedI / Asp718 digested pHE4a expression vector.

2. Construction of VEGF-2 T103-R227 at pHE4

Amplified Directed Polymerase Chain Reaction and E. To allow sub-cloning of VEGF-2 T103-R227 (amino acids 103-227 in FIG. 1 or SEQ ID NO: 18) into E. coli protein expression vector pHE4, complementary to the desired region of VEGF-2, having the following base sequence: Synthesize two oligonucleotide primers:

5 'primer (coding sequences of NdeI / START and 18nt):

5'-GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3 '(SEQ ID NO: 19)

3 ′ primer (coding sequences of Asp718, STOP, and 15nt):

5'-GCA GCA GGT ACC TCA ACG TCT AAT AAT GGA-3 '(SEQ ID NO: 21).

In the case of the aforementioned primers, the NedI or Asp718 restriction sites are incorporated into the 5 'primer and the 3' primer, respectively. The 5 'primer (SEQ ID NO: 19) is also known as E. coli. The 3 ′ primer (SEQ ID NO: 21) contains the ATG sequence adjacent to the VEGF-2 coding region and within the same frame, allowing translation of the cloned fragment in E. coli. It contains one stop codon (preferably used in E. coli) in the same frame adjacent to the VEGF-2 coding region, which ensures correct translation termination in E. coli.

The polymerase chain reaction is carried out using standard conditions well known to those skilled in the art and using the nucleotide sequence of the mature VEGF-2 (amino acids 24 to 419 in SEQ ID NO: 18) as a template, as constructed in Example 3. . The resulting amplicon is subjected to restriction digestion with NedI and Asp718 and subcloned into the NedI / Asp718 digested pHE4a expression vector.

3. Construction of VEGF-2 T103-L215 in pA2GP

In this example, the plasmid shuttle vector vector pA2GP, using a baculovirus leader and standard methods described in the following, was used to determine the N-terminal and C-terminal deleted VEGF-2 proteins (see FIG. 1 or SEQ ID NO: 18). Cloned DNA encoding amino acids 103-215 is used to insert into baculovirus to express such N-terminal and C-terminal deleted proteins. Summers et al., A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). Such expression vectors are potent polyhedrin promoters of Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by secretion signal peptides (leaders) of the baculovirus gp67 protein, and BamHI, XbaI and Asp718. Contain such convenient restriction sites. The polyadenylation site of monkey virus 40 (“SV 40”) is used for efficient polyadenylation. For easy selection of recombinant viruses, E. coli. Beta-galactosidase from E. coli is contained in the same orientation under the control of a weak drosophila promoter followed by the polyadenylation signal of the polyhedrin gene. The inserted gene is flanked by viral sequences on both sides for cell-mediated homologous recombination with wild-type viral DNA, resulting in live cells expressing the cloned polynucleotides.

Many other baculovirus vectors, such as pAc373, pVL941 and pAc1M1, provide as long as the construct provides a properly localized signal for transcription, translation, secretion, etc., eg, signal peptide and AUG in frame as needed. May be used instead of the vector. Such vectors are described, for example, in Luckow, V.A. and Summers, M.D., Virology, 170: 31-39 (1989).

In FIG. 1, the cDNA sequence encoding the VEGF-2 protein without 102 amino acids at the N-terminus and 204 amino acids at the C-terminus is amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. Let's do it.

The 5 'primer has the sequence 5'-GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA-3' (SEQ ID NO: 22) and is intended to stay in the same frame as the BamHl restriction enzyme site (in bold) following the vector-supplied signal peptide One spacer nt and the coding sequence 17nt of the VEGF-2 protein. The 3 'primer has the sequence 5N-GCA GCA TCT AGA TCA CAG TTT AGA CAT GCA-3' (SEQ ID NO: 23) and complements the XbaI restriction site (in bold) and the 3 'coding sequence of the stop codon and VEGF-2. Contains dog nucleotides.

Amplified sequences are separated from 1% agarose gels using a commercial kit (trade name: “Geneclean” from BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with endonuclease BamHI and XbaI and purified again on 1% agarose gel. This fragment is linked to the pA2GP baculovirus transfer vector (Supplier) at the BamHI and XbaI sites. Through this linkage, VEGF-2 cDNAs (amino acids 103-215 in FIG. 1 or SEQ ID NO: 18) representing N-terminal and C-terminal deleted VEGF-2 proteins are in the same frame as the signal sequence of the baculovirus GP gene. Cloned and located at the 3 'end of the signal sequence in the vector. It is named pA2GPVEGF-2.T103-L215.

4. Construction of VEGF-2 T103-R227 in pA2GP

In FIG. 1, the cDNA sequence encoding the VEGF-2 protein without 102 amino acids at the N-terminus and 192 amino acids at the C-terminus (ie, amino acids 103 to 227 of SEQ ID NO: 18) is the 5 'and 3' sequence of the gene. Amplify using the corresponding PCR oligonucleotide primers.

5'-GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA ATT TGC TGC-3 'primers were followed by BamH restriction site (in bold) followed by 1 spacer nt, and VEGF to stay in the same frame as the vector-supplied signal peptide -2 has a sequence (SEQ ID NO: 24) containing 26 nt encoding base of the protein. The 3 'primer has the sequence 5N-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3' (SEQ ID NO: 25) and complements the XbaI restriction site (in bold) and the 3 'coding sequence of the stop codon and VEGF-2. Contains 21 nucleotides.

Amplified sequences are separated from 1% agarose gels using a commercial kit (trade name: “Geneclean” from BIO 101 Inc., La Jolla, Ca.). The fragment is then digested with endonuclease BamHI and XbaI and purified again on 1% agarose gel. This fragment is linked to the pA2GP baculovirus transfer vector (Supplier) at the BamHI and XbaI sites. Through this linkage, the VEGF-2 cDNA (amino acids 103-227 in FIG. 1 or SEQ ID NO: 18) representing the N-terminal and C-terminal deleted VEGF-2 proteins is in the same frame as the signal sequence of the baculovirus GP gene. Cloned and located at the 3 'end of the signal sequence in the vector. It is named pA2GPVEGF-2.T103-R227.

5. Construction of VEGF-2 in pC1

Expression vectors pC1 and pC4 are potent promoters (LTR) plus CMV enhancer of the Raus sarcoma virus [Cullen et al., Molecular and Cellular Biology, 438-447 (3 March 1985)] (Boshart et al., Cell 41: 521- 530 (1985). For example, multiple cloning sites with restriction enzyme cleavage sites BamHI, XbaI and Asp718 facilitate cloning of the gene of interest. Such vectors contain, in addition to the 3N intron, polyadenylation and termination signals of the preproinsulin gene.

Vector pC1 is used for the expression of VEGF-2 protein. Plasmid pC1 is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids contain the mouse DHFR gene under the control of the SV40 early promoter. The cells were selected by growing Chinese hamster ovary- or other cells lacking dihydrofolate activity transfected with these plasmids in a selection medium supplemented with chemotherapeutic methotrexate (alpha minus MEM, Life Technologies). Can be. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) is well known in the art. Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Biol. Chem. 253: 1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097: 107-143, Page, M.J. and Sydenham, M. A. 1991, Biotechnology 9: 64-68. Cells grown at increasing concentrations of MTX result in amplification of the DHFR gene, resulting in drug resistance by overproducing the target enzyme DHFR. When the second gene is linked to this DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this approach can be used to generate cell lines carrying more than 1,000 copies of amplified gene (s). Subsequently, when the administration of methotrexate is stopped, the cell line contains the amplified gene integrated into the chromosome (s).

The plasmid vector pC4 is a long terminal repeat (LTR) plus human megalovirus (CMV) of the potent promoter of the Raus sarcoma virus (Cullen et al., Molecular and Cellular Biology, 438-447) for expression of the gene of interest [Boshart et al., Cell 41: 521-530 (1985), which contain fragments isolated from enhancers of the immediate early genes. The bottom of this promoter is the following single restriction enzyme cleavage site that allows integration of the genes: BamHI, PvuII, and Nrul. Outside these cloning sites, the plasmid contains the translational stop codein in all three reading frames, followed by the polyadenylation site of the 3N intron and the rat preproinsulin gene. Other high efficient promoters such as the human b-actin promoter, the SV40 early or late promoter, or other retroviruses such as long terminal repeats from HIV and HTLVI may also be used for the expression. For polyadenylation of mRNA, for example, human growth hormone or other signals from globin genes can also be used.

Stable cell lines carrying genes of interest integrated into the chromosome can also be selected upon cotransfection with selectable markers such as gpt, G418 or hygromycin. It is advantageous to use one or more screening markers at the outset, for example G418 plus methotrexate.

Plasmid pC1 is digested with restriction enzyme BamHI and then dephosphorylated using calf phosphate by procedures known in the art. The vector is then separated from the 1% agarose gel.

DNA sequence encoding VEGF-2 (ATCC Accession No. 97149) is constructed by PCR using the following two primers corresponding to the 5 'and 3' ends of the VEGF-2 gene: 5 'primer (5 '-GAT CGA TCC ATC ATG CAC TCG CTG GGC TTC TTC TCT GTG GCG TGT TCT CTG CTC G-3' (SEQ ID NO: 26)) encodes a VEGF-2 coding sequence 40nt starting from the cleno-filled BamHI site and initiation codon. Contains; The 3 'primer (5'-GCA GGG TAC GGA TCC TAG ATT AGC TCA TTT GTG GTC TTT-3' (SEQ ID NO: 27)) contains the BamHI site and 16 nt of the VEGF-2 coding sequence without stop codon.

PCR amplified fragments as above are separated from the 1% agarose gel as mentioned above, then digested with endonuclease BamHI and purified again on the 1% agarose gel. The isolated fragments and the dephosphorylated vector are linked using T4 DNA ligase. Then, this. E. coli HB101 cells are transformed and bacteria containing plasmid pC1 are identified. The sequence and orientation of such inserted genes is confirmed by DNA sequencing. This construct is named pC1VEGF-2.

6. Construction of pC4SigVEGF-2 T103-L215

Plasmid pC4Sig is plasmid pC4 (Accession No. 209646) that contains a protein signal sequence as well as a human IgG Fc portion.

In order to allow amplification directed polymerase chain reaction and sub-cloning of VEGF-2 T103-L215 (amino acids 103-215 in FIG. 1 or SEQ ID NO: 18) into pC4Sig, the desired VEGF-2 has the following base sequence: Synthesize two oligonucleotide primers complementary to the region:

5 'primer (coding sequence of BamHI and 26nt):

5'-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3 '(SEQ ID NO: 34)

3 'primer (coding sequence of XbaI, STOP, and 15nt):

5'-CGT CGT TCT AGA TCA CAG TTT AGA CAT GCA TCG GCA G-3 '(SEQ ID NO: 35).

The polymerase chain reaction is carried out using standard conditions well known to those skilled in the art and using the nucleotide sequence (amino acids 24 to 419) of mature VEGF-2 as constructed in Example 3 as a template. The resulting amplicons are restriction digested with BamHI and XbaI and subcloned into BamHI / XbaI digested pC4Sig vector.

7. Construction of pC4SigVEGF-2 T103-R227

In order to allow amplification directed polymerase chain reaction and sub-cloning of VEGF-2 T103-L215 (amino acids 103-227 in FIG. 1 or SEQ ID NO: 18) into pC4Sig, the desired VEGF-2 has the following base sequence: Synthesize two oligonucleotide primers complementary to the region:

5 'primer (coding sequence of BamHI and 26nt):

5'-GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3 '(SEQ ID NO: 34)

3 ′ primer (coding sequences of XbaI, STOP, and 21nt):

5'-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3 '(SEQ ID NO: 25).

The polymerase chain reaction is carried out using standard conditions well known to those skilled in the art and using the nucleotide sequence (amino acids 24 to 419) of mature VEGF-2 as constructed in Example 3 as a template. The resulting amplicons are restriction digested with BamHI and XbaI and subcloned into BamHI / XbaI digested pC4Sig vector.

8. Construction of pC4VEGF-2 M1-M263

Plasmid vector pC4 is a potent promoter (LTR) plus CMV-enhancer of Laus' sarcoma virus (Cullen et al., Molecular and Cellular Biology, 438-447 (1985. 3.)) [Boshart et al., Cell 41: 521-530 (1985). For example, multiple cloning sites with restriction enzyme cleavage sites BamHI, XbaI and Asp718 facilitate cloning of the gene of interest. Such vectors contain, in addition to the 3N intron, polyadenylation and termination signals of the rat preproinsulin gene.

In this example, cloned DNA (amino acids 1 to 263 in FIG. 1 or SEQ ID NO: 1) encoding the C-terminal deleted VEGF-2 M1-M263 protein was inserted into the plasmid vector pC4 to delete C-terminal VEGF. -2 expresses protein.

To allow amplification-directed polymerase chain reaction and sub-cloning of VEGF-2 M1-M263 into the expression vector pC4, two oligonucleotide primers complementary to the desired region of VEGF-2, having the following base sequence, were synthesized: do:

5 'primer: 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3' (SEQ ID NO: 28)

3 'primer: 5'-GAC TGG TAC CTT ATC ACA TAA AAT CTT CCT GAG CC-3' (SEQ ID NO: 29).

For the 5 'primers mentioned above, the BamHI restriction site is incorporated, whereas for the 3' primers, the Asp718 restriction site is incorporated. The 5 ′ primer also contains 6nt, 20nt of VEGF-2 coding sequence, and E. coli. While the 3 'primer contains an ATG sequence adjacent to the VEGF-2 coding region and within the same frame, which allows translation of the cloned fragment in E. coli, the 3' primer contains 2nt, 20nt of VEGF-2 coding sequence, and E. coli. It contains one stop codon (preferably used in E. coli) in the same frame adjacent to the VEGF-2 coding region, which ensures correct translation termination in E. coli.

The polymerase chain reaction is carried out using standard conditions well known to those skilled in the art and using the nucleotide sequence (amino acids 24 to 419) of mature VEGF-2 as constructed in Example 3 as a template. The resulting amplicon is subjected to restriction digestion with BamHI and Asp718 and subcloned into BamHI / Asp718 digested pC4 protein expression vector. This construct is named pC4VEGF-2 M1-M263.

9. Construction of pC4VEGF-2 M1-M311

In this example, cloned DNA (amino acids 1 to 311 in FIG. 1 or SEQ ID NO: 1) encoding the C-terminal deleted VEGF-2 M1-M311 protein was inserted into the plasmid vector pC4 and the C-terminal deleted VEGF. -2 expresses protein.

To allow amplification-directed polymerase chain reaction and sub-cloning of VEGF-2 M1-M311 into the expression vector pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 with the following base sequence were synthesized: do:

5 'Primer: 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3' (SEQ ID NO: 30)

3 'primer: 5'-GAC TGG TAC CTT ATC AGT CTA GTT CTT TGT GGG G-3' (SEQ ID NO: 31).

For the 5 'primers mentioned above, the BamHI restriction site is incorporated, whereas for the 3' primers, the Asp718 restriction site is incorporated. The 5 ′ primer also contains 6nt, 20nt of VEGF-2 coding sequence, and E. coli. While the 3 'primer contains an ATG sequence adjacent to the VEGF-2 coding region and within the same frame, which allows translation of the cloned fragment in E. coli, the 3' primer contains 2nt, 20nt of VEGF-2 coding sequence, and E. coli. It contains one stop codon (preferably used in E. coli) in the same frame adjacent to the VEGF-2 coding region, which ensures correct translation termination in E. coli.

The polymerase chain reaction is carried out using standard conditions well known to those skilled in the art and using the nucleotide sequence (amino acids 24 to 419) of mature VEGF-2 as constructed in Example 3 as a template. The resulting amplicon is subjected to restriction digestion with BamHI and Asp718 and subcloned into BamHI / Asp718 digested pC4 protein expression vector.

10. Construction of pC4VEGF-2 M1-Q367

In this example, cloned DNA (amino acids 1 to 311 in FIG. 1 or SEQ ID NO: 1) encoding the C-terminal deleted VEGF-2 M1-D311 protein was inserted into the plasmid vector pC4 and the C-terminal deleted VEGF. -2 expresses protein.

To allow amplification-directed polymerase chain reaction and sub-cloning of VEGF-2 M1-D311 into the expression vector pC4, two oligonucleotide primers complementary to the desired region of VEGF-2, having the following base sequence, were synthesized: do:

5 'primer: 5'-GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3' (SEQ ID NO: 32)

3 'primer: 5'-GAC TGG TAC CTC ATT ACT GTG GAC TTT CTG TAC ATT C-3' (SEQ ID NO: 33).

For the 5 'primers mentioned above, the BamHI restriction site is incorporated, whereas for the 3' primers, the Asp718 restriction site is incorporated. The 5 ′ primer also contains 6nt, 20nt of VEGF-2 coding sequence, and E. coli. While the 3 'primer contains an ATG sequence adjacent to the VEGF-2 coding region and within the same frame, which allows translation of the cloned fragment in E. coli, the 3' primer contains 2nt, 20nt of VEGF-2 coding sequence, and E. coli. It contains one stop codon (preferably used in E. coli) in the same frame adjacent to the VEGF-2 coding region, which ensures correct translation termination in E. coli.

The polymerase chain reaction is carried out using standard conditions well known to those skilled in the art and using the nucleotide sequence (amino acids 24 to 419) of mature VEGF-2 as constructed in Example 3 as a template. The resulting amplicon is subjected to restriction digestion with BamHI and Asp718 and subcloned into BamHI / Asp718 digested pC4 protein expression vector. This construct is named pC4VEGF-2 M1-Q367.

Example 10 Transient Expression of VEGF-2 Protein in COS-7 Cells

Experimental design

Expression of the VEGF-2-HA fusion protein from the construct prepared in Example 4 is described, for example, in Harlow et al .; Antibodies: A Laboratory Manual, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)] for detection by radiolabeling and immunoprecipitation. To this end, two days after transfection, cells are labeled for 8 hours by culturing in medium containing 35S-cysteine. Cells and media were then collected and cells were washed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, et al. , See above, using the HA specific monoclonal antibodies to precipitate proteins from both cell melt and culture medium The precipitated proteins are analyzed by SDS-PAGE and autoradiography.

result

As shown in FIG. 16, cells transfected with pcDNA1 VEGF-2HA secrete 56kd and 30kd proteins. 56kd protein but not 30kd protein is also detected in the cell melt, but not in the control group. This suggests that the 30kd protein probably came from cleavage of the 56kd protein. Since the HA-tag is on the C-terminus of VEGF-2, the 30 kd protein should represent the C-terminal portion of the truncated protein, whereas the N-terminal portion of the truncated protein cannot be detected by immunoprecipitation. These data indicate that the VEGF-2 protein expressed in mammalian cells is secreted and processed.

Example 11 Stimulation Effect of VEGF-2 on Proliferation of Vascular Endothelial Cells

Experimental design

Expression of VEGF-2 is abundant in highly vascularized tissues. Thus, the role of VEGF-2 in regulating the proliferation of several types of endothelial cells is examined.

Endothelial Cell Proliferation Assay

To assess the mitotic activity of growth factors, colorimetric MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3- using an electron coupling reagent PMS (phenazine methosulfate) Carboxymethoxyphenyl) -2- (4-sulfophenyl) 2H-tetrazolium) assay is performed (CellTiter 96 AQ, Promega). Cells are seeded (5,000 cells / well) in 96-well plates in 0.1 ml serum-supplemented medium and attached overnight. After serum-depletion in 0.5% PBS for 12 hours, conditions with or without heparin (8 U / ml) (bFGF, VEGF ₁₆₅ or VEGF-2 in 0.5% FBS) are added to the wells for 48 hours. 20 mg of MTS / PMS mixture (1: 0.05) is added per well and incubated for 1 hour at 37 ° C. before absorbance at 490 nm is measured in an ELISA plate reader. The background absorbance from control wells (slight medium, no cells) is reduced and seven wells are performed in parallel with each condition. Leak et al. In Vitro Cell. Dev. Biol. 30A: 512-518 (1994).

result

VEGF-2 slightly propagated human umbilical vein endothelial cells (HUVEC) and intradermal microvascular cells (FIGS. 17 and 18). This stimulatory effect of VEGF-2 is more pronounced for the proliferation of endometrial and microvascular endothelial cells (FIG. 19). Endometrial endothelial cells (HEEC) responded positively to VEGF-2 (96% of the effect of VEGF-2 on microvascular endothelial cells). Half of the microvascular endothelial cells (HMEC) against VEGF-2 are 73% compared to VEGF-2. The response of HUVEC and BAEC (bovine aortic endothelial cells) to VEGF-2 is substantially as low as 10% and 7%, respectively. The activity of VEGF-2 protein differs between different purification runs, and the stimulatory effect of certain batches on HUVEC proliferation is significantly higher than that of other batches.

Example 12 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation

VEGF-2 expression is high in vascular smooth muscle cells. Smooth muscle is an important therapeutic target for vascular diseases such as restenosis. To assess the potential effect of VEGF-2 on smooth muscle cells, the effect of VEGF-2 on human aortic smooth muscle cell (HAoSMC) proliferation is examined.

Experimental design

HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly stated, subconfluent, resting cells grown on 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. The cells are then pulsed with 10% calf serum and 6 mg / ml BrdUrd. After 24 hours, immunocytochemistry is performed by using BrdUrd staining kit (Zymed Laboratories). Briefly, the cells are incubated with biotinylated mouse anti-BrdUrd antibody at 4 ° C. for 2 hours after exposure to denaturing solution, followed by incubation with streptavidin-peroxidase and diaminobenzidine. After backstaining with hematoxylin, the cells are placed for microscopic examination and BrdUrd-positive cells are counted. BrdUrd index is calculated as the ratio of BrdUrd-positive cells to total cell number. In addition, simultaneous detection of BrdUrd staining (nuclei) and FITC uptake (cytoplasm) are performed on individual cells by simultaneously using bright room illumination and dark-UV fluorescent illumination. Hayashida et al., J. Biol. Chem. 6: 271 (36): 21985-21992 (1996).

result

VEGF-2 has an inhibitory effect on PDGF-induced vascular smooth muscle cell proliferation, but no inhibitory effect on that induced by fetal bovine serum (FBS).

Example 13: Stimulation of Endothelial Cell Migration

Endothelial cell migration is an important step associated with angiogenesis.

Experimental design

This example will be used to test the possibility that VEGF-2 can stimulate lymphoid endothelial cell migration. At present, there are no published reports on this model. However, we will essentially adopt a model of vascular endothelial cell migration for use as lymphoid endothelial cells as follows:

Endothelial cell migration assays are performed using a 48 well microchemotactic chamber. Neuroprobe Inc., Cabin John, MD; Falk, W., Goodwin, RHJ, and Leonard, EJ "A 48 well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration", J. Immunological Methods 1980; 33: 239-247]. A polyvinylpyrrolidone-free polycarbonate filter (Nucleopore Corp. Cambridge, Mass.) With a pore size of 8 μm is coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. The test substance is diluted to the appropriate concentration in M199 supplemented with 0.25% bovine serum albumin (BSA) and 25 μl of the final dilution is placed in the lower chamber of the modified Bodene apparatus. Subconfluent, initial passage (2-6) HUVEC or BMEC cultures are washed and treated with trypsin for the minimum amount of time necessary to achieve cell detachment. After placing the filter between the lower and upper chambers, 2.5 × 10 ⁵ cells suspended in 50 μl M199 containing 1% FBS are seeded into the upper compartment. The device is then incubated for 5 hours at 37 ° C. in a hygroscopic room with 5% CO 2 to move cells. After the incubation period, the filter is removed and the top portion of the filter with unmoved cells is scraped with a rubber polyman. The filter is fixed with methanol and stained with Gemsa solution (Diff-Quick, Baxter, McGraw Park, IL). Migration is quantified by counting three random high-voltage (40x) cells in each well and performed four times for all groups.

result

In assays that examine HUVEC migration using a 43-well microchemotaxis chamber, VEGF-2 can stimulate migration of HUVECs (see FIG. 21).

Example 14 Stimulation of Nitric Oxide by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelial relaxation. VEGF-1 has been shown to induce nitric oxide production by endothelial cells in response to VEGF-1. As a result, VEGF-2 activity can be assayed by determining nitric oxide production by endothelial cells in response to VEGF-2.

Experimental design

After 24 hours depletion and subsequent 4 hours of exposure to various levels of VEGF-1 and VEGF-2, nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells. Nitric oxide in the medium is determined by using a Greiss reagent that measures total nitrite after reduction of nitric oxide-induced nitrate by nitrate reductase. The effect of VEGF-2 on nitric oxide release is examined on HUVEC.

Briefly stated, NO release from cultured HUVEC monolayers is measured using a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) 1049. Calibration of the NO element is performed according to the following equation:

2KNO ₂ + 2KI + 2H ₂ SO ₄ 6 2NO + I ₂ + 2H ₂ O + 2K ₂ SO ₄

A standard calibration curve is obtained by adding graded concentrations of KNO ₂ (0.5, 10, 25, 50, 100, 250 and 500 nmol / L) to a calibration solution containing KI and H ₂ SO ₄ . The specificity of the Iso-NO electrode to NO is determined in advance by measuring NO from the original NO gas 1050. The culture medium is removed and the HUVEC is washed twice with Dulbecto phosphate buffered saline. The cells are then immersed in 5 ml of filtered Krebs-Hanzelite solution in 6-well plates and the cell plates are maintained on a slide warmer (Lab Line Instruments Inc.). To maintain the temperature at 37 ° C. Before applying different conditions, the NO sensor probe is inserted perpendicular to the well, keeping the top of the 2 mm electrode under the surface of the solution. S-nitro acetyl penicillamine (SNAP) is added as a positive control. The amount of NO released is expressed as picomolar per 1 × 10 ⁶ endothelial cells. All values reported are the average of 4 to 6 measurements in each group (number of cell culture wells). Leak et al. Biochem. and Biophys. Res. Comm. 217: 96-105 (1995).

result

VEGF-2 can stimulate nitric oxide release to HUVEC (FIG. 22) to higher levels than VEGF. This suggests that VEGF-2 can modify vascular permeability and vasodilation.

Example 15 Effect of VEGF-2 on Ligament Formation in Angiogenesis

Another step in angiogenesis is ligament formation characterized by the differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (pupillary structures) when cultured in vitro.

Experimental design

CADMEC (microvascular endothelial cells) are purchased as proliferative (passage 2) cells from Cell Applications, Inc., cultured in CADMEC growth medium for cell applications and used in passage 5. For the in vitro angiogenesis assay, wells of 48-well cell culture plates are coated with adhesion factor medium (200 ml / well) of cell application at 37 ° C. for 30 minutes. CADMEC is seeded at 7,500 cells / well in the coated wells and incubated overnight in growth medium. Subsequently, the growth medium is replaced with ligament formation medium of 300 mg cell application containing control buffer or HGS protein (0.1-100 ng / ml), and the cells are further incubated for 48 hours. The number and length of capillary-like bands are quantified using a Beckerler VIA-170 video imaging analyzer. All assays are performed three times.

Commercial (R & D) VEGF (50 ng / ml) is used as a positive control. b-esteradiol (1 ng / ml) is used as a negative control. Appropriate buffers (containing no protein) are also used as controls.

result

VEGF-2 was observed to inhibit ligament formation similarly to IFNa, which also stimulates endothelial cell proliferation (FIG. 23). This inhibitory effect may be a secondary effect of endothelial proliferation which is mutually excluded with the ligament formation process.

Example 16: Angiogenesis Effects on Chick Villi

Chick choriourema (CAM) is a well established system for testing for angiogenesis. Angiogenesis on CAM is readily visible and quantifiable. Examine the ability of VEGF-2 to stimulate angiogenesis in CAM.

Experimental design

Embryo

Fertilized eggs of White Leghorn chick (Gallus gallus) and Japanese Koturnix coturnix are incubated at 37.8 ° C. and 80% humidity. Differentiated CAMs of 16-day-old chicks and 13-day guar embryos are studied in the following manner.

CAM black

On day 4 of development, a spear is made into the eggshell of the chick egg. Embryos are examined for normal development and the eggs are sealed with cello tape. They are further cultured by 13 days. Dermanox coverslips (Nunc, Naperville, IL) are cut into discs about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and pipet about 3.3 mg / 5 ml onto the disc. After air drying, the displaced disk is applied to the CAM. After 3 days, the supernatant is fixed in 3% glutaraldehyde and 2% formaldehyde and washed in 0.12 M sodium cacodylate buffer. These are photographed with stereomicroscope [Wild M8] and enclosed for semi- and ultratin sections as described below. Control experiments are performed using only carrier discs.

result

This data demonstrates that VEGF-2 stimulates angiogenesis nine times as compared to the untreated control in the CAM assay. However, this stimulus is only 45% of the VEGF stimulus level (FIG. 24).

Example 17 Angiogenesis Assay Using Matrigel Implantation in Mice

Experimental design

Methylcellulose disks containing either 20 mg of BSA (negative control) and 1 mg of bFGF and 0.5 mg of VEGF-1 (positive control) to establish an in vivo model for angiogenesis to test protein activity. Were implanted subcutaneously in mice and rats.

Although BSA discs seemed to show little angiogenesis, positive control discs signaled angiogenesis. On day 9, one mouse showed a clear response to bFGF.

result

All of the VEGF-2 proteins are believed to have enhanced Matrigel cellularity by about two factors by visual evaluation.

Thirty more mice were transplanted with BSA, bFGF, and disks containing various amounts of VEGF-1, VEGF-2-B8, and VEGF-2-C4. Instead of one control disc and one experimental disc, two identical discs are implanted in each mouse.

Samples of all recovered disks are immunostained with von Willebrand factor to detect the presence of endothelial cells in the disk and stained with flk-1 and flk-4 to distinguish between vascular endothelial cells and lymphoid endothelial cells. . However, limited histochemical analysis of neovascularization and lymphoid angiogenesis could not be determined.

Example 18 Ischemic Recovery in the Rabbit Lower Limb Model

Experimental design

To study the in vivo effect of VEGF-2 on ischemia, a rabbit Fuji ischemia model is created by surgically removing one femoral artery as mentioned previously. Takeshita, S. et al., Am . J. Pathol 147: 1649-1660 (1995). Due to the incision of the femoral artery, the thrombosis degenerates and the external iliac artery is blocked. As a result, blood flow to the ischemic limb depends on secondary blood vessels originating from the internal iliac arteries. Takeshita, S. et al., Am. J. Pathol 147: 1649-1660 (1995). It takes 10 days for the rabbit to recover after surgery and for the growth of endogenous collateral vessels. Ten days after surgery (day 0), after performing baseline angiograms, 500 mg of ischemic limb internal iliac artery was exposed by arterial gene transfer technique using a hydrogel-coated balun catheter as described in Transfected VEGF-2 expressing plasmids. See Riessen, R. et al. Hum Gene Ther. 4: 749-758 (1993); Leclerc, G. et al. J. Clin. Invest. 90: 936-944 (1992). When VEGF-2 is used for treatment, a single bolus of 500 mg VEGF-2 protein or control is delivered via the infusion catheter into the internal iliac artery of the ischemic limb over one minute. On day 30, various parameters are measured in these rabbits.

result

Both VEGF-2 protein (FIG. 25, top panel) and exposed expression plasmids (FIG. 25, middle panel) can restore the following parameters in ischemic paper. Restoration of blood flow, angiograph scores appears to be somewhat more by administering a 500 mg plasmid compared to the case with 500 mg protein (FIG. 25, bottom panel). The degree of recovery is comparable to that by VEGF in separate experiments (data not shown). Vasodilators cannot achieve the same effect, suggesting that blood flow restoration is not simple due to vasodilation effects.

a. BP Ratios (FIG. 25A)

Pressure ratio of contractile pressure of ischemic limb to normal limb contractile.

2. Bloodstream and Bloodstream Preservation (FIG. 25B)

Resting FL: Blood Flow During Unexpanded Conditions

Maximum FL: blood flow during fully expanded ecology (also indirect measurement of angioplasty)

Blood flow conservation is reflected as maximum FL: resting FL.

3. Angiograph Score (FIG. 25C)

This is measured by the angiogram of the ancillary vessels. Crossover turbid arteries are used to determine the score as the ratio of circles in the overlaying grid divided by the total number m in the rabbit thighs.

4. Capillary Density (FIG. 25D)

The number of incidental capillaries was determined in light microscopic sections taken from Fuji.

As discussed, VEGF-2 is processed into co-purified N-terminal and C-terminal fragments. Such N-terminal fragments contain original putative functional regions and may be involved in biological activity.

Example 19: Effect of VEGF-2 on Vasodilation

As mentioned above, VEGF-2 can stimulate NO release, a mediator of vascular endothelial dilatation. Because vascular endothelial dilation is important for lowering blood pressure, we examine the ability of VEGF-2 to affect blood pressure in spontaneous hypertensive rats (SHR). VEGF-2 caused a dose dependent decrease in diastolic blood pressure (FIGS. 26A and B). Increasing the dose of VEGF-2 steadily lowered diastolic blood pressure, and statistical significance was obtained when the 300 mg / kg dose was administered. The change observed at this dose is no different than that observed with acetylcholine (0.5 mg / kg). Lower average arterial pressure (MAP) was also observed (FIGS. 26C and d). VEGF-2 (300 mg / kg) and acetylcholine lowered the MAP of these SHR animals to normal.

Additionally, increasing doses of B8, C5 and C4 preps of VEGF-2 (0, 10, 30, 100, 300 and 900 mg / kg) are administered to 13-14 week old spontaneous hypertensive rats (SHR). Data are shown as mean ± SEM. Statistical analysis is performed with a paired t-test and defines statistical significance as response to p <0.05 versus buffer alone.

Studies with VEGF-2 (C5 prep) have shown that this significantly lowers blood pressure, but the response is not as great as with VEGF-2 (B8 prep) even at doses of 900 mg / kg. appear.

Studies with VEGF-2 (C4 preparation) showed that CHO expressed protein preparations produced results similar to those observed using C5 (ie, with statistical significance, rather than that observed with B8 preparations). Much less) (FIGS. 26A-D).

Experiments are performed with another CHO-expressed protein M-CIF, as a control and the C4 and C5 batches of VEGF-2 resulted in minor but statistically significant blood pressure changes. When M-CIF was administered at doses ranging from 10 to 900 mg / kg, no significant change in diastolic blood pressure occurred. A slight statistically significant decrease in mean arterial blood pressure was observed at doses of 100 to 900 mg / kg, but no dose responsiveness was observed. These results suggest that the blood pressure drop observed using the C4 and C5 batches of VEGF-2 is specific, ie VEGF-2 related.

Example 20 Rat Ischemic Skin Flap Model

Experimental design

Evaluation parameters include cutaneous blood flow, skin temperature and factor VIII immunohistochemistry or endothelial alkaline phosphatase responses. VEGF-2 expression during cutaneous ischemia is studied using in situ hybridization.

The study in this model is divided into three parts:

a) ischemic skin

b) ischemic skin trauma

c) normal trauma.

Experimental protocols include:

a) Growing a single diameter sufficient-thickness random skin flap (muscle flap throughout the animal's lower abdomen) of 3x4 cm.

b) Incisional cuts in ischemic skin (4-6 mm in diameter).

c) Topical treatment of incisional wounds with VEGF-2 (day 0, 1, 2, 3, 4 after wound): 1 mg to 100 mg at various dosage ranges as follows.

d) Wound tissues were harvested at 3, 5, 7, 10, 14 and 21 days after wounding for histological, immunohistochemistry and in situ studies.

Example 21 Peripheral Arterial Disease Model

Angiogenesis therapy with VEGF-2 has been developed as a novel therapy for restoring the tongue around ischemia in the case of peripheral arterial disease.

Experimental design

Experimental protocols include:

a) connects one side of the femoral artery to produce ischemic muscle of Fuji, the other side of Fuji acts as a control.

b) VEGF-2 protein in doses ranging from 20 mg to 500 mg is delivered intravenously and / or intramuscularly three times weekly for 2-3 weeks.

c) Ischemic muscle tissue is collected after ligation of the femoral arteries at week 1, 2 and 3 to analyze VEGF-2 expression and histology. Biopsies are also performed on the other side of normal muscle of the contralateral Fuji.

Example 22 Ischemic Myocardial Disease Model

VEGF-2 is evaluated as a potent mitotic substance that can stimulate collateral vessel growth and reconstruct new vessels after coronary artery occlusion. Changes in VEGF-2 expression are examined in situ.

Experimental design

Experimental protocols include:

a) The heart is exposed through left thoracotomy in rats. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the rib cage is closed.

b) VEGF-2 protein in doses ranging from 20 mg to 500 mg is delivered intravenously and / or intramuscularly three (perhaps) three times per week for two to four weeks.

c) 30 days after surgery, the heart is taken out, cross-sectioned for body shape and analyzed in situ.

Example 23 Rat Corneal Wound Healing Model

This animal model shows the effect of VEGF-2 on angiogenesis.

Experimental design

Experimental protocols include:

a) A 1 to 1.5 mm long incision is made from the corneal center into the interstitial layer.

b) Insert the tongue out under the lip of the incision facing the outer corner of the eye.

c) make pockets (the basis of which is 1 to 1.5 mm around the eyes).

d) In the pocket, the pellet containing 50-500 mg VEGF-2 is placed.

e) VEGF-2 treatment may also be administered topically to the corneal wound at a dose of 20 to 500 mg (daily dose for 5 days).

Example 24 Diabetic Mice and Glucocorticoid-damaged Wound Healing Models

Experimental design

Experimental protocols include:

1. Diabetic db + / db + mouse model

To demonstrate that VEGF-2 promotes the healing process, a genetic diabetic mouse model of wound healing is used. The wound healing model of sufficient thickness in the db + / db + mouse model is a widely identified and clinically relevant regeneration model of damaged wound healing. Healing of diabetic wounds relies on the formation and re-epithelialization of granulated tissue rather than contraction. Gartner, M.H. et al., J. Surg. Res. 52: 389 (1992); Greenhalgh, D.G. et al. Am. J. Pathol. 136: 1235 (1990).

Diabetic animals have many characteristic features observed in type II diabetes. Homozygous (db + / db +) mice are obese compared to their normal heterozygous (db + / + m) compatriots. Mutant diabetic (db + / db +) mice have a single autosomal recessive mutation for chromosome 4 (db +). Coleman et al. Proc. Natl. Acad. Sci. USA 77: 283-293 (1992). Animals exhibit polyphagia, polyhydramnios and polyuria. Mutant diabetic mice (db + / db +) had elevated blood glucose levels, increased or normal insulin levels, and cell-mediated immunity was inhibited. Mandel et al., J. Immunol. 120: 1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51 (1): 1-7 (1983); Leiter et al., Am. J. of Pathol. 114: 46-55 (1985). Peripheral neuropathy, myocardial complications and microvascular lesions, basement membrane hypertrophy and glomerular filtration abnormalities have been described in these animals. Norido, F. et al., Exp. Neurol. 83 (2): 221-232 (1984); Robertson et al., Diabetes 29 (1): 60-67 (1980); Giacomelli et al., Lab Invest. 40 (4): 460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl): 1-6 (1982). These homozygous diabetic mice exhibit hyperglycemia that is resistant to insulin similar to human type II diabetes. Mandel et al., J. Immunol. 120: 1375-1377 (1978).

The characteristics observed in these animals suggest that the healing in this model may be similar to the healing observed in human diabetes. Greenhalgh, et al., Am. J. of Pathol. 136: 1235-1246 (1990).

animal

Genetic diabetic female C57BL / KsJ ((db + / db +) mice and their non-diabetic ((db + / + m) heterozygous compatriots are used in this study (Jackson Laboratories). Purchase 6- and 8-year-olds Breed the animals individually and provide sufficient food and water Use sterile techniques to perform all manipulations Human Genome Science Institutional Animal Care and Use Committe Carry out all experiments in accordance with the rules and guidelines of the laboratory and the care and use of laboratory animals.

Surgery Creation

The wound creation protocol is performed according to the previously reported method. Tsuboi, R. and Rifkin, D.B., J. Exp. Med. 172: 245-251 (1990). Briefly stated, animals are anesthetized by intraperitoneal injection on the day of wound creation with avertin (0.01 mg / ml), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. . The back area of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical site is dried with sterile gauze before wound formation. Subsequently, a 8 mm thick wound is made using a Kayes tissue punch. Immediately after creating the wound, the surrounding tissue is carefully scraped off to prevent the wound from expanding. This wound is left exposed for the duration of the experiment. Topical treatment for 5 consecutive days beginning on the day of wound creation. Prior to treatment, the wound is carefully cleaned with sterile saline and gauze sponges.

The wound is visually inspected and photographed on the day of surgery and at a fixed distance two days thereafter. Wound closure is determined by daily measurements on days 1-5. Wounds are measured horizontally and vertically using graduated Jameson calipers. If the granulated tissue is no longer visible and the wound site is covered with continuous epithelium, the wound is considered to heal.

VEGF-2 is administered using different doses of VEGF-2 in the vehicle, ranging from 4 to 500 mg per wound per day for 8 days. The vehicle control group received 50 ml of vehicle solution.

Animals are euthanized on day 8 with intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound area and surrounding tissue are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Experimental design

Three groups of 10 animals each (5 diabetes and 5 non-diabetic controls) are evaluated: 1) vehicle placebo control, 2) VEGF-2.

Wound Area and Suture Measurement

Wound closure is analyzed by measuring areas within the vertical and horizontal axes and obtaining the total wound area. The shrinkage is then assessed by establishing the difference between the initial wound area (day 0) and the post-treatment wound area (day 8). The wound area on day 1 is 64 mm 2, which corresponds to the intradermal punch size. Calculate using the following equation:

[8th open area]-[1st open area] / [1st open area]

histology

Samples are fixed in 10% buffered formalin and paraffin-embedded blocks are divided at right angles to the wound surface (5 mm) and cut using a Reichert-Yun microtome. Conventional hematoxylin-eosin (H & E) staining is performed on the cross-sectional area of the dividing wound. Histological examination of the wound is used to assess whether the healing process and the morphological appearance of the repaired tissue has been modified by treatment with KGF-2. Such assays include demonstrating cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epithelial maturation. See Greenhalgh, D.G. et al., Am. J. Pathol. 136: 1235 (1990). A graduated lens micrometer is used by the blind observer.

Immunohistochemistry

Re-epithelialization

Tissue sections are immunohistochemically stained with polyclonal rabbit anti-human keratin antibodies using the ABC Ellite detection system. Human skin is used as a positive tissue control, while non-immune IgG is used as a negative control. Keratinocyte growth is determined by assessing the extent of wound re-epithelialization using a graduated lens micrometer.

Cell proliferation marker

Proliferative cell nuclear antigen / cyclin (PCNA) in skin samples is demonstrated by using anti-PCNA antibodies (1:50) using the ABC Ellite detection system. Human colon cancer is used as a positive tissue control and human brain tissue is used as a negative tissue control. Each sample contains fragments with the primary antibody omitted and substitutions with non-immune mouse IgG. Grading these sections ranges from 0 to 8, based on the extent of proliferation, with low grades reflecting some growth and high grades reflecting intensive growth.

Statistical analysis

Analyze experimental data using the unpaired t test. A p value of <0.05 is considered significant.

B. Steroid Damaged Rat Models

Inhibition of wound healing by steroids has been widely demonstrated in various in vitro and in vivo systems. See Wahl, S.M. Glucocorticoids and Wound healing. In: Anti-Imflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahl, S.M. et al., J. Immunol. 115: 476-481 (1975); Werb, Z. et al., J. Exp. Med. 147: 1684-1694 (1978). Glucocorticoids increase angiogenesis and vascular permeability [Ebert, R. H., et al., An. Intern. Med. 37: 701-705 (1952)], fibroblast proliferation and collagen synthesis (Beck, L.S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B.F. et al., J. Clin. Invest. 61: 703-797 (1978)] and by causing a temporary decrease in circulating monocytes (see Haynes, B.F., et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, S.M., "Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)] delay wound healing. Systemic administration of steroids to damaged wound healing is a well established phenomenon in rats. Beck, L.S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, S.M., "Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce, G. F., et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989).

To demonstrate that VEGF-2 can promote the healing process, the effect of multiple topical administration of VEGF-2 on incised skin wounds of sufficient thickness in rats whose healing has been compromised by systemic administration of methylprednisolone is evaluated.

animal

Young but mature male Sprague Dawley rats (Charles River Laboratories) weighing 250-300 g are used in this experiment. At the beginning of the study, these animals are purchased at 8 and 9 week olds. The healing response is impaired by systemic administration of methylprednisolone (17 mg / kg rat muscle) at wound formation. Breed animals individually and provide plenty of food and water. All operations are performed using aseptic technique. All experiments are performed in accordance with the rules and guidelines of the Institutional Animal Care and Use Committe and the protection and use of laboratory animals.

Surgery Creation

The wound creation protocol is performed according to section A above. On day 0 of wound formation, animals are anesthetized by intraperitoneal injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The back area of the animal is shaved and the skin is washed with 70% ethanol solution and iodine solution. The surgical site is dried with sterile gauze before wound formation. Subsequently, a 8 mm thick wound is made using a Kayes tissue punch. This wound is left exposed for the duration of the experiment. The test substance is administered topically for 7 consecutive days starting on the day of wound creation followed by methylprednisolone. Prior to treatment, the wound is carefully cleaned with sterile saline and gauze sponges.

Visually inspect the wound and take pictures at a fixed distance on the day of surgery and on the last day of treatment. Wound closure is determined by daily measurements on days 1-5. Wounds are measured horizontally and vertically using graduated Jameson calipers. If the granulated tissue is no longer visible and the wound site is covered with continuous epithelium, the wound is considered to heal.

VEGF-2 is administered using different doses of VEGF-2 in the vehicle, ranging from 4 to 500 mg per wound per day for 8 days. The vehicle control group received 50 ml of vehicle solution.

Animals are euthanized on day 8 with intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound area and surrounding tissue are then harvested for histological examination. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Experimental design

Four groups of 10 animals each (five were methylprednisolone administered and five glucocorticoids administered) were evaluated: 1) untreated group, 2) vehicle placebo control, 3) VEGF-2 treated group.

Wound Area and Suture Measurement

Wound closure is analyzed by measuring areas within the vertical and horizontal axes and obtaining the total wound area. The suture is then assessed by establishing the difference between the initial wound area (day 0) and the post-treatment wound area (day 8). The wound area on day 1 is 64 mm 2, which corresponds to the intradermal punch size. Calculate using the following equation:

[8th open area]-[1st open area] / [1st open area]

histology

Samples are fixed in 10% buffered formalin and the paraffin-embedded blocks are divided at right angles to the wound surface (5 mm) and cut using an Olympus microtome. Conventional hematoxylin-eosin (H & E) staining is performed on the cross-sectional area of the dividing wound. Histological examination of the wound is used to assess whether the healing process and morphological appearance of the repaired tissue has been improved by treatment with VEGF-2. A graduated lens micrometer is used by the blind observer to determine the wound gap distance.

Statistical analysis

Analyze experimental data using the unpaired t test. A p value of <0.05 is considered significant.

Example 25 Specific Peptide Fragments for Producing VEGF-2 Monoclonal Antibodies

Four specific peptides (named SP-40, SP-41, SP-42 and SP-43) are generated. This will be used to generate monoclonal antibodies for analyzing VEGF-2 processing. This peptide is shown below:

1. "SP-40": MTVLYPEYWKMY (amino acids 70 to 81 in SEQ ID NO: 18)

2. "SP-41": KSIDNEWRKTQSMPREV (amino acids 120 to 136 in SEQ ID NO: 18 (see C-> S mutation at 131))

3. "SP-42": MSKLDVYRQVHSIIRR (amino acids 212 to 227 in SEQ ID NO: 18)

4. "SP-43": MFSSDAGDDSTDGFHDI (amino acids 263 to 279 in SEQ ID NO: 18)

Example 26 Lymphoma Animal Model

The purpose of this experimental approach is to generate an appropriate and consistent lymphoma model for testing the resetting of the lymphatic circulation in rats and the therapeutic effect of VEGF-2 in lymphatic angiogenesis. Efficacy is measured by swelling volume of ill Fuji, quantification of lymphatic vasculature amount, total plasma protein and histopathology. Acute lymphoma is observed for 7-10 days. Perhaps more importantly, chronic progression of edema is accompanied by 3-4 weeks.

Experiment process

Prior to starting surgery, a blood sample is taken for protein concentration analysis. Pentobarbital is administered to male rats weighing approximately 350 g. In succession, the right leg is shaved from knee to hip. This shaved area is wiped off with gauze immersed in 70% EtOH. Blood is taken for serum total protein testing. After marking two measurement levels (0.5 cm above the heel in the middle of the back foot), circumferential and volumetric measurements are performed prior to injection of the dye into the foot. Inject the 0.05 ml of 1% Evans Blue into the intradermal back of the right and left feet. Circumference and volumetric measurements are then performed after the dye is injected into the feet.

Using the knee joint as a marker, the mid-leg inguinal incision is circumferentially allowing localization of the femoral vessels. Forceps and hemostatic forceps are used to dissect and separate the skin flap. After localizing the femoral vessels, localized lymphatic vessels are performed along the sides and below the vessels. The major lymphatic vessels in this area are then electrically coagulated or sutured.

Using a microscope, the muscles of the back of the leg (near the tendons and the adductor muscle) are incised smoothly. Localization of the tendon muscle lymph node. The original blood supply of the two near and two distant lymphatic vessels and the groin muscle lymph node is then connected by sutures. The subchondral lymph nodes and all accompanying adipose tissue are removed by cutting the connective tissue.

Care should be taken to control any weak bleeding resulting from this process. After blocking the lymph cells, the skin plate is sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin edges are sealed with the underlying muscle tissue, creating a 0.5 cm gap around the leg. If desired, the skin can be directed to it by suturing the lower muscles.

To avoid infection, the animals are screened (no bedding) and raised individually. Animals that are recovered are checked daily through an optimal edema peak, which typically occurs on days 5-7. The flat edema peak is then observed. To assess the intensity of lymphedema, we measured the circumference and volume of two named batches on each foot daily for seven days prior to surgery. Plasma proteins have an effect on lymphedema and determine whether protein analysis is a useful test parameter. The weight of both control and edema was evaluated in 2 batches. The analysis is performed in a blind manner.

Circumferential measurement: Under simple gas anesthesia to prevent finger movements, the circumference is measured using cloth tape. Measurements are made at the ankle bone and dorsal foot by two different persons and the two readings are averaged. Readings are taken from the control and edema.

Volumetric Measurements: On the day of surgery, animals are anesthetized and tested with pentobarbital prior to surgery. For the daily volume, animals are simply halotan anesthetized (quick immobilization and rapid recovery), both legs shaved, and the same markings using waterproof markers on the legs. The legs are first immersed in water and then immersed into the instrument at each marked level and then measured by Buco edema software (Chen / Victor). Data is recorded from one person while the other is immersed in the marked area.

Plasma Protein Determination: Blood is drawn, radiated and serum isolated prior to surgery and concluded for total protein and Ca2 + comparison.

Fat weight comparison: After taking blood, the animal is prepared for tissue collection. The paper is cut using quiltine, and then the experimental and control legs are cut at the joint and weighed. A second weighing is performed as the Tibio-Canyl joint is articulated and the feet weighed.

Histological preparations: The area of transverse muscle (slaughter muscle) located behind the knee is cut, arranged in a metal mold filled with cryogel, immersed in cold methylbutane and placed in a labeled bag at -80EC until sectioned. . Upon sectioning, the muscles are observed under fluorescence microscopy against lymphatic cells. Other immunohistochemical methods are currently being evaluated.

Example 27: Method of Treatment Using Gene Therapy for Production of VEGF-2 Polypeptides in Vivo

Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases and conditions. This method of gene therapy relates to the introduction of exposed nucleic acids (DNA, RNA and antisense DNA or RNA) comprising VEGF-2 operably linked to a promoter to increase expression of VEGF-2. Such gene therapy and delivery techniques and methods are known in the art. See WO 90/11092, WO 98/11779; US Patents 5693622, 55155151, 5580859; Tabata H. et al. (1997) Cardivasc. Res. 35 (3): 470-479, Chao, J. et al. (1997) Pharmacol. Res. 35 (6): 517-522, Wolff, J.A. (1997) Neuromuscul. Disord. 7 (5): 314-318, Schwarz, B. et al., (1996) Gene Ther. 3 (5): 405-411, Tsurumi, Y. et al. (1996) Circulation 94 (12): 3281-3290; Incorporated herein by reference.

The VEGF-2 polynucleotide construct may be delivered by any method of delivering an injectable substance to cells of the animal, for example, by injection into tissues of the interstitial region of the heart (heart, muscle, skin, lung, liver, intestine, etc.). Can be. The VEGF-2 polynucleotide construct may also be delivered directly to the artery. The VEGF-2 polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “exposed” polynucleotide, DNA, or RNA refers to any transport vehicle that acts to assist, enhance or promote the introduction into cells, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents, and the like. Refers to sequences that do not have. However, VEGF-2 polynucleotides can be prepared by methods well known to those skilled in the art [Felgner P.L. et al. (1995) Ann. NY Acad. Sci. 772: 126-139 and Abdallah B. et al. (1995) Biol. Such as taught in Cell 85 (1): 1-7.

The VEGF-2 vector constructs used in gene therapy methods are preferably constructs that do not integrate into the host genome or contain no sequences that permit replication. Unlike other gene therapy techniques, one major advantage of introducing exposed nucleic acid sequences into target cells is the temporaryity of polynucleotide synthesis in the cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to produce the desired polypeptide for a period of up to six months.

VEGF-2 constructs include muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testes, ovaries, uterus, rectum, It can be transported to the interstitial spaces of tissues in animals, including the nervous system, eyes, lines and connective tissue. The interstitial spaces of such tissues include mucopolysaccharide matrices of intercellular fluid, elastic fibers in blood vessel or chamber walls, collagen fibers of fibrous tissue, or cattle in connective tissue or bone covering muscle cells, among the reticular fibers of organ tissue. It contains the same matrix within. Similarly, lymphatic fluid of spatial and lymphatic channels occupied by circulating plasma is included. They can be conveniently delivered by injection into tissues comprising these cells. They are preferably achieved in cells that are transported and expressed in persistent non-differentiating cells to be differentiated, but which are not differentiated or less fully differentiated, for example, liver cells or blood fibroblasts of blood. Can be. Preferably, they are delivered by direct injection into the artery.

For exposed polynucleotide injections, effective dosages of DNA or RNA will range from about 0.05 to about 50 mg / kg body weight. The dosage will preferably be about 0.005 to about 20 mg / kg body weight, more preferably about 0.05 to about 5 mg / kg body weight. Of course, as will be appreciated by those skilled in the art, such dosage will vary depending on the tissue site being injected. Appropriate and effective dosages of nucleic acid sequences can be readily determined by those skilled in the art and can depend on the disease to be treated and the route of administration. Preferred routes of administration are parenteral injection routes into the interstitial space of tissues, or direct injection routes into arteries. However, other parenteral routes may also be used, such as inhalation of an aerosol formulation specifically for delivery to lung or bronchial tissue, throat or nasal mucosa. In addition, the exposed VEGF-2 construct may be delivered to the artery during angioplasty by the catheter used in the procedure.

The dose responsive effect of the injected VEGF-2 polynucleotide construct in artery in vivo is determined as follows: Template DNA suitable for producing mRNA encoding VEGF-2 is prepared according to standard recombinant DNA methods. Template DNA, which may be circular or linear, is used as exposed DNA or complexes with liposomes. Subsequently, various amounts of template DNA are injected into the rabbit artery.

Fuji ischemia in rabbits is induced using surgery, as described in Example 18. Immediately after this was performed, 3 ml syringes and 2-gauge needles prepared through small skin incisions were applied to five different sites of the adductor muscle (2 sites), medialage (2 sites) and semi-membrane muscles (1 site). Direct injection of plasmid DNA encoding VEGF-2.

Treating ischemia by measuring the number of capillaries in light microscopic sections taken from treated Fuji, measuring calf blood pressure, and measuring blood flow rate in the arterial Doppler guidewire compared to ischemic Fuji from untreated rabbits. Determine capacity. Takeshita et al., J. Clin. Invest. 93: 662-670 (1994). Using the results of these experiments in rabbits, VEGF-2 polynucleotide exposed DNA can be used to extrapolate appropriate and other therapeutic parameters from humans and other animals.

Example 28 Treatment Methods Using Gene Therapy In Vitro

One gene therapy method is implanting fibroblasts capable of expressing a VEGF-2 polypeptide onto a patient. Generally, fibroblasts are obtained from a skin biopsy subject. The resulting tissue is placed in tissue-culture medium and separated into small pieces. A small piece of tissue is placed on the wet surface of the tissue culture flask and approximately 10 pieces are placed in each flask. The flask is rotated up and down, tightly sealed and left overnight at room temperature. After 24 hours at room temperature, the flask is inverted and the tissue pieces are fixed at the bottom of the flask and fresh medium (eg, Hans F12 medium supplemented with 10% PBS, penicillin and streptomycin) is added. The flask is then incubated at 37 ° C. for approximately one week.

At this time, fresh medium is added and changed continuously every few days. After two more weeks of incubation, fibroblast monolayers appear. This monolayer is trypsinized and weighed into a larger flask.

PMV-7 flanked by long terminal repeats of Moroni murine sarcoma virus, Kirschmeier, P.T. et al, DNA, 7: 219-25 (1988)] are digested with Eco RI and HindIII and subsequently treated with calf intestinal phosphatase. Linear vectors are fractionated on agarose gels and purified using glass beads.

CDNA encoding VEGF-2 is amplified using PCR primers corresponding to the 5 'and 3' terminal sequences, respectively, as shown in Example 1. Preferably, the 5 'primer contains an EcoRI site and the 3' primer comprises a HindIII site. Equal amounts of Moroni murine sarcoma virus linear extension and amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for linking the two fragments. This linkage mixture is used to transform bacterial HB101 and then plated onto kanamycin-containing agar for the purpose of confirming that the vector contains VEGF-2 with the appropriate insertion.

Amphoric pA317 or GP + am12 packaging cells are grown in tissue culture to confluence density in Dulbecco's Modified Eagle's Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. Subsequently, an MSV vector containing the VEGF-2 gene is added to the medium and packaging cells are transduced with the vector. The packaging cells then produce infectious viral particles containing the VEGF-2 gene (such packaging cells are referred to as producer cells).

Fresh medium is added to the transduced producer cells, and the medium is subsequently harvested from 10 cm plates of confluent producer cells. The spent medium containing infectious virus particles is filtered through a Millipore filter to remove detached producer cells. This medium is then used to infect fibroblast cells. The medium is removed from the sub-confluent plate of fibroblasts and quickly replaced with medium from producer cells. Remove this medium and replace with fresh medium. If the titer of the virus is high, practically all fibroblasts will be infected and no screening is required. If this titer is very low, it may be necessary to use retroviral vectors with selectable markers such as neo or his. Once fibroblasts are efficiently infected, the fibroblasts are analyzed to determine if VEGF-2 protein is produced.

Genetically engineered fibroblasts are then injected alone into the host, or after growth with confluence on cytodex 3 microcarrier beads.

Example 29: Method of Treatment Using Gene Therapy Homologous Recombination

Another gene therapy method according to the present invention involves operably linking an endogenous VEGF-2 sequence with a promoter via homologous recombination as described in US Pat. No. 5,641,670 (June 24, 1997). .); International Publication No. WO 96/29411 (September 26, 1996); International Publication No. WO 94/12650 (August 4, 1994); Koller et al., Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); and Zijlstra et al., Nature 342: 435-438 (1989). Such methods include activating genes that are present in the target cell but are not normally expressed in the cell or are expressed at levels below the desired level.

A polynucleotide construct containing a promoter and a targeting sequence homologous to the 5 'non-coding sequence of endogenous VEGF-2, flanking the promoter is made. Since the targeting sequence is sufficiently near the 5 'end of VEGF-2, this promoter will be operably linked to the endogenous sequence upon homologous recombination. Promoter and targeting sequences can be amplified using PCR. Preferably, such amplified promoters contain separate restriction enzyme sites on the 5 'and 3' ends. Preferably, the 3 'end of the first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second targeting sequence is the same restriction enzyme site as the 3' end of the amplified promoter It contains.

The promoter and targeting sequences thus amplified are digested with appropriate restriction enzymes and then treated with calf intestinal phosphatase. The promoter thus digested and the digested targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for linking the two fragments. The construct is size fractionated on agarose gel and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as an exposed polynucleotide via electroporation. However, such polynucleotide constructs may also be administered with transfection promoters, such as liposomes, viral sequences, viral particles, precipitants, and the like. Such methods of transport are known in the art.

Once the cells are infected, homologous recombination will occur, resulting in a promoter that is operably linked to the endogenous VEGF-2 sequence. As a result, VEGF-2 is expressed in the cells. Expression can be detected by immunological scanning or other methods known in the art.

Fibroblasts are obtained from a biopsy subject. The resulting tissue is placed in DMEM + 10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are treated with trapsin and washed from plastic surfaces with nutrient media. For counting, an equal volume of cell suspension is removed and the remaining cells are centrifuged. The supernatant is degassed and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are recentrifuged, the supernatant is degassed, and the cells are resuspended in electroporation buffer containing serum albumin of 1 mg / ml acetylated bovine. The final cell suspension contains approximately 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. To construct a plasmid targeting the VEGF-2 site, plasmid pUC18 (MBI Fermentas, Amherst, NY) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site at the 5 'end and a BamHI site at the 3' end. Amplify two VEGF-2 non-coding sequences via PCR: one VEGF-2 non-coding sequence (VEGF-2 fragment 1) is amplified by HindIII at the 5 'end and amplified by the Xba site at the 3' end; The other VEGF-2 non-coding sequence (VEGF-2 fragment 2) is amplified with BamHI at the 5 'end and a HindIII site at the 3' end. CMV promoter and VEGF-2 fragments are digested with appropriate enzymes (CMV promoter-XbaI and BamHI; VEGF-2 fragment 1-XbaI; VEGF-2 fragment 2-BamHI) and linked together. The resulting linkage product is digested with HindIII and linked with HindIII-digested pUC18 plasmid.

Plasmid DNA is added to sterile cuvettes with 0.4 cm electrode gap (Bio-Rad). Final DNA concentration is generally at least 120 μg / ml. Then 0.5 ml of cell suspension (containing approximately 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and the DNA solution are gently mixed. Electroporation is performed using a Gin-Pulse device (Bio-Rad). Set the capacitance and voltage to 960μF and 250-300V, respectively. As the voltage increases, cell viability decreases, but the proportion of viable cells that stably incorporate introduced DNA into their genome increases significantly. If these parameters are given, a pulse time of about 14-20 mSec should be observed.

The electroporated cells are maintained at room temperature for about 5 minutes and the components of the cuvette are gently removed with a sterile transfer pipette. The cells are added directly to pre-warmed nutrient medium (DMEM with 15% calf serum) in 10 cm dishes and incubated at 37EC. The next day, the medium is degassed, replaced with 10 ml of fresh medium and incubated for another 16 to 24 hours.

Genetically engineered fibroblasts are then injected alone into the host, or after growth with confluence on cytodex 3 microcarrier beads. These fibroblasts then produce protein products.

Example 30 VEGF-2 Transgenic Animals

VEGF-2 polypeptides may also be expressed in transgenic animals. Any, including but not limited to mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheeps, cows and non-human primates such as baluns, monkeys, and chimpanzees Species of animals may also be used to generate transgenic animals. In certain embodiments, the polypeptides of the invention are expressed in humans as part of a gene therapy protocol using techniques described herein or methods known in the art.

All techniques known in the art are used to introduce transformed genes (ie, polynucleotides of the invention) into an animal to create a founder line of transgenic animals. Such techniques include pro-nuclear microinjection [Paterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., US Pat. No. 4,873,191 (1989); Retroviral mediated gene transfer into the germline [Van der Putten et al., Proc. Natl. Acad. Sci. USA 82: 6148-6152 (1985); undifferentiated embryonic cells or embryos; Gene targeting in embryonic liver cells (Thomson et al., Cell 56: 313-321 (1989)); Electroporation of cells or embryos. Lo, 1983, Mol Cell. Biol. 3: 1803-1814 (1983); Introduction of polynucleotides of the invention using gene guns (Ulmer et al., Science 259: 1745 (1993)); Introducing a nucleic acid construct into an embryonic pleuripotent liver cell and retranslating such liver cell back into undifferentiated embryonic cells; And sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989)), and the like. For a review of these techniques, reference may be made to Gordon, "Transgenic Animals", Intl. Rev. Cytol. 115: 171-229 (1989), incorporated herein by this reference.

All techniques known in the art, for example, transgenic clones containing polynucleotides of the present invention, using nuclear phase transfer of nuclei from cultured embryo, fetal or adult cells induced in resting phase to nucleated oocytes. Can be produced by Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997).

The present invention provides not only transgenic animals carrying genes transformed in all of their cells, but also mosaic animals or chimeric animals carrying genes transformed in some but not all cells. Such transformed genes may be integrated as single transformed genes or as multiple copies, such as in concatama, for example as head-to-head tandem or head-to-tail tandem. Such transformed genes are also described in Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992), which can be selectively introduced into and activated in specific cell types. The regulatory sequences required for such cell type specific activation will depend on the particular cell type of interest and will be apparent to those skilled in the art. Gene targeting is preferred when the polynucleotide transformed gene is desired to be integrated into the chromosomal region of the endogenous gene.

Briefly, when using this technique, a vector containing several nucleotide sequences homologous to an endogenous gene is designed for the purpose of integrating through homologous recombination having a chromosomal sequence and disrupting the function of the nucleotide sequence of the endogenous gene. By selectively introducing the transformed gene into specific cell types [see; Gu et al., Science 265: 103-106 (1994)] can inactivate endogenous genes. The regulatory sequences required for such cell type specific inactivation will depend on the particular cell type of interest and will be apparent to those skilled in the art.

Once the transgenic animal is generated, expression of the recombinant gene can be assayed using standard techniques. Initial screening is accomplished by Southern blot analysis or PCR techniques to analyze the tissue to see if integration of the transformed genes has been achieved. Expression levels of mRNA of transformed genes in tissues of transgenic animals include, but are not limited to, Northern blot analysis, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR) of tissue samples obtained from animals. Can be evaluated using Using antibodies specific for the transformed gene product, a sample of tissue expressing the transformed gene can be immunocytochemically or immunohistochemically assessed.

Once founder animals are produced, they can be crossed, co-breeding, bi-crossing or cross-crossing to produce colonies of specific animals. Examples of such breeding strategies include crossbreeding founder animals with one or more integration sites to establish separate lines; Syngeneic separate lines to generate compound transforms that express higher levels of the transformed gene due to the additional expression effect of each transformed gene; Cross-crossing heterozygous transgenic animals to produce animals that are homozygous for a given integration site to enhance expression and eliminate the need for screening of animals by DNA analysis; Crossing separate homozygous lines to create compound heterozygous or homozygous lines; And mating to place the transformed gene on a separate background suitable for the experimental model of interest.

The transgenic animals of the present invention are useful for screening for compounds that are effective in making biological functions of VEGF-2 polypeptides, studying conditions and / or disorders associated with abnormal VEGF-2 expression, and alleviating such conditions and / or disorders. Its uses include but are not limited to model systems.

Example 31 VEGF-2 Knock-Out Animals

Endogenous VEGF-2 gene expression can also be lowered by inactivating or "knocking out" the VEGF-2 gene and / or its promoter using targeted homologous recombination. Smithies et al., Nature 317: 230 -234 (1985); Thomas & Capecchi, Cell 51: 503-512 (1987); Thompson et al. Cell 5: 313-321 (1989); Incorporated herein by reference. For example, a non-functional polynucleotide (or a completely unrelated DNA sequence) which is a mutant of the invention flanked by DNA homologous to an endogenous polynucleotide sequence (coding region or regulatory region of the gene), Cells that express the polypeptide of the invention in vivo can be used with or without a selectable marker and / or a negative selectable marker. In another embodiment, knockouts are generated in cells containing, but not expressing, genes of interest using techniques known in the art. Insertion of the DNA construct via targeted homologous recombination inactivates the targeted gene. This approach is particularly suitable for research and agricultural applications where modifications to embryonic liver cells are used to generate animal progeny with inactive targeting genes (see Thomas & Capecchi 1987 and Thomas 1989, supra). However, this approach can be adapted for use in humans when the recombinant DNA constructs have been administered or targeted to the necessary site in vivo using appropriate viral vectors that will be apparent to those skilled in the art.

In a further aspect of the invention, cells (knockouts) that have been genetically treated to express the polypeptides of the invention or genetically treated to express the polypeptides of the invention are administered to the patient in vivo. Such cells can be obtained from the patient (ie, animals including humans) or MHC miscible donors, which include fibroblasts, bone marrow cells, blood cells (eg white blood cells), adipocytes, muscle cells, endothelial cells, etc. May be included, but is not limited thereto. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequences of the polypeptides of the invention into the cells or to use plasmids, cosmids, YACs, exposed DNA, electroporation, liposomes. Coding sequences associated with the polypeptides of the invention and / or by transduction (using a viral vector, preferably a vector that integrates the transformed gene into the cell genome) or transfection process, including but not limited to Endogenous regulatory sequences are disrupted. The coding sequence of a polypeptide of the invention can be placed under the control of a potent construct or inducible promoter or promoter / enhancer to achieve expression and preferably secretion of a VEGF-2 polypeptide. Genetically treated cells that express and preferably secrete the polypeptides of the invention can be systemically introduced into the patient, eg, circulated or intraperitoneally.

Alternatively, the cells can be incorporated into the matrix and transplanted into the body, eg, genetically engineered fibroblasts can be implanted as part of a skin graft; Genetically treated endothelial cells can be implanted as part of a lymphatic or vascular graft. Anderson et al., US Pat. No. 5,339,349; and Mulligan & Wilson, US Pat. No. 5,460 959; Incorporated herein by reference.

If the cells to be administered are non-magnetic or non-MHC compatible cells, they can be administered using well known techniques for inhibiting the progression of the host immune response against the introduced cells. For example, the cells can be introduced in encapsulated form, allowing the introduced cells to be recognized by the host immune system, while immediately exchanging the components to the extracellular environment.

Knockout animals of the invention make animal models useful for screening compounds that are effective in making biological functions of VEGF-2 polypeptides, studying conditions and / or disorders associated with abnormal VEGF-2 expression, and alleviating such conditions and / or disorders. Its uses include, but are not limited to, systems.

Many modifications and variations of the present invention are possible in light of the above teachings, and therefore, the invention may be practiced unless specifically stated otherwise.

The entirety of all publications cited herein (including patents, patent applications, journals, laboratory manuals, books, or other printed matter) are incorporated herein by reference.

<110> HUMAN GENOME SCIENCES, INC.

<120> Vascular endothelial growth factor 2

<130> 5-1998-084740-7

<150> US

<151> 1998-03-13

<150> US

<151> 1998-06-30

<160> 35

<170> KOPATIN 1.5

<210> 1

<211> 1674

<212> DNA

<213> Artificial Sequence

<220>

<223> the full length nucletide sequence of VEGF-2

<220>

<221> sig_peptide

<222> (12) .. (80)

<220>

<221> mat_peptide

<222> (81) .. (1268)

<220>

<221> CDS

<222> (12) .. (1268)

<400> 1

GTCCTTCCAC C ATG CAC TCG CTG GGC TTC TTC TCT GTG GCG TGT TCT CTG 50

Met His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu

-23 -20 -15

CTC GCC GCT GCG CTG CTC CCG GGT CCT CGC GAG GCG CCC GCC GCC GCC 98

Leu Ala Ala Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala

-10 -5 1 5

GCC GCC TTC GAG TCC GGA CTC GAC CTC TCG GAC GCG GAG CCC GAC GCG 146

Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala

10 15 20

GGC GAG GCC ACG GCT TAT GCA AGC AAA GAT CTG GAG GAG CAG TTA CGG 194

Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg

25 30 35

TCT GTG TCC AGT GTA GAT GAA CTC ATG ACT GTA CTC TAC CCA GAA TAT 242

Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr

40 45 50

TGG AAA ATG TAC AAG TGT CAG CTA AGG AAA GGA GGC TGG CAA CAT AAC 290

Trp Lys Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn

55 60 65 70

AGA GAA CAG GCC AAC CTC AAC TCA AGG ACA GAA GAG ACT ATA AAA TTT 338

Arg Glu Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe

75 80 85

GCT GCA GCA CAT TAT AAT ACA GAG ATC TTG AAA AGT ATT GAT AAT GAG 386

Ala Ala Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu

90 95 100

TGG AGA AAG ACT CAA TGC ATG CCA CGG GAG GTG TGT ATA GAT GTG GGG 434

Trp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly

105 110 115

AAG GAG TTT GGA GTC GCG ACA AAC ACC TTC TTT AAA CCT CCA TGT GTG 482

Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val

120 125 130

TCC GTC TAC AGA TGT GGG GGT TGC TGC AAT AGT GAG GGG CTG CAG TGC 530

Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys

135 140 145 150

ATG AAC ACC AGC ACG AGC TAC CTC AGC AAG ACG TTA TTT GAA ATT ACA 578

Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr

155 160 165

GTG CCT CTC TCT CAA GGC CCC AAA CCA GTA ACA ATC AGT TTT GCC AAT 626

Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn

170 175 180

CAC ACT TCC TGC CGA TGC ATG TCT AAA CTG GAT GTT TAC AGA CAA GTT 674

His Thr Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val

185 190 195

CAT TCC ATT ATT AGA CGT TCC CTG CCA GCA ACA CTA CCA CAG TGT CAG 722

His Ser Ile Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln

200 205 210

GCA GCG AAC AAG ACC TGC CCC ACC AAT TAC ATG TGG AAT AAT CAC ATC 770

Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile

215 220 225 230

TGC AGA TGC CTG GCT CAG GAA GAT TTT ATG TTT TCC TCG GAT GCT GGA 818

Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly

235 240 245

GAT GAC TCA ACA GAT GGA TTC CAT GAC ATC TGT GGA CCA AAC AAG GAG 866

Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu

250 255 260

CTG GAT GAA GAG ACC TGT CAG TGT GTC TGC AGA GCG GGG CTT CGG CCT 914

Leu Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro

265 270 275

GCC AGC TGT GGA CCC CAC AAA GAA CTA GAC AGA AAC TCA TGC CAG TGT 962

Ala Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys

280 285 290

GTC TGT AAA AAC AAA CTC TTC CCC AGC CAA TGT GGG GCC AAC CGA GAA 1010

Val Cys Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu

295 300 305 310

TTT GAT GAA AAC ACA TGC CAG TGT GTA TGT AAA AGA ACC TGC CCC AGA 1058

Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg

315 320 325

AAT CAA CCC CTA AAT CCT GGA AAA TGT GCC TGT GAA TGT ACA GAA AGT 1106

Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser

330 335 340

CCA CAG AAA TGC TTG TTA AAA GGA AAG AAG TTC CAC CAC CAA ACA TGC 1154

Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys

345 350 355

AGC TGT TAC AGA CGG CCA TGT ACG AAC CGC CAG AAG GCT TGT GAG CCA 1202

Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro

360 365 370

GGA TTT TCA TAT AGT GAA GAA GTG TGT CGT TGT GTC CCT TCA TAT TGG 1250

Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp

375 380 385 390

CAA AGA CCA CAA ATG AGC TAAGATTGTA CTGTTTTCCA GTTCATCGAT 1298

Gln Arg Pro Gln Met Ser

395

TTTCTATTAT GGAAAACTGT GTTGCCACAG TAGAACTGTC TGTGAACAGA GAGACCCTTG 1358

TGGGTCCATG CTAACAAAGA CAAAAGTCTG TCTTTCCTGA ACCATGTGGA TAACTTTACA 1418

GAAATGGACT GGAGCTCATC TGCAAAAGGC CTCTTGTAAA GACTGGTTTT CTGCCAATGA 1478

CCAAACAGCC AAGATTTTCC TCTTGTGATT TCTTTAAAAG AATGACTATA TAATTTATTT 1538

CCACTAAAAA TATTGTTTCT GCATTCATTT TTATAGCAAC AACAATTGGT AAAACTCACT 1598

GTGATCAATA TTTTTATATC ATGCAAAATA TGTTTAAAAT AAAATGAAAA TTGTATTTAT 1658

AAAAAAAAAA AAAAAA 1674

<210> 2

<211> 419

<212> PRT

<213> Artificial Sequence

<220>

<223> the deduced amino acid sequence of VEGF-2

<400> 2

Met His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala

-23 -20 -15 -10

Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe

-5 1 5

Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala

10 15 20 25

Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser

30 35 40

Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met

45 50 55

Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln

60 65 70

Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala

75 80 85

His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys

90 95 100 105

Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe

110 115 120

Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr

125 130 135

Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr

140 145 150

Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu

155 160 165

Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser

170 175 180 185

Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile

190 195 200

Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn

205 210 215

Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys

220 225 230

Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser

235 240 245

Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu

250 255 260 265

Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys

270 275 280

Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys

285 290 295

Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu

300 305 310

Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro

315 320 325

Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys

330 335 340 345

Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr

350 355 360

Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser

365 370 375

Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro

380 385 390

Gln met ser

395

<210> 3

<211> 1526

<212> DNA

<213> Artificial Sequence

<220>

<223> the nucleotide sequence of a truncated, biologically active form

of VEGF-2

<220>

<221> sig_peptide

<222> (71) .. (142)

<220>

<221> mat_peptide

<222> (143) .. (1120)

<220>

<221> CDS

<222> (71) .. (1120)

<400> 3

CGAGGCCACG GCTTATGCAA GCAAAGATCT GGAGGAGCAG TTACGGTCTG TGTCCAGTGT 60

AGATGAACTC ATG ACT GTA CTC TAC CCA GAA TAT TGG AAA ATG TAC AAG 109

Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys

-24 -20 -15

TGT CAG CTA AGG AAA GGA GGC TGG CAA CAT AAC AGA GAA CAG GCC AAC 157

Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asn

-10 -5 1 5

CTC AAC TCA AGG ACA GAA GAG ACT ATA AAA TTT GCT GCA GCA CAT TAT 205

Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr

10 15 20

AAT ACA GAG ATC TTG AAA AGT ATT GAT AAT GAG TGG AGA AAG ACT CAA 253

Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln

25 30 35

TGC ATG CCA CGG GAG GTG TGT ATA GAT GTG GGG AAG GAG TTT GGA GTC 301

Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val

40 45 50

GCG ACA AAC ACC TTC TTT AAA CCT CCA TGT GTG TCC GTC TAC AGA TGT 349

Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys

55 60 65

GGG GGT TGC TGC AAT AGT GAG GGG CTG CAG TGC ATG AAC ACC AGC ACG 397

Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr

70 75 80 85

AGC TAC CTC AGC AAG ACG TTA TTT GAA ATT ACA GTG CCT CTC TCT CAA 445

Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln

90 95 100

GGC CCC AAA CCA GTA ACA ATC AGT TTT GCC AAT CAC ACT TCC TGC CGA 493

Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg

105 110 115

TGC ATG TCT AAA CTG GAT GTT TAC AGA CAA GTT CAT TCC ATT ATT AGA 541

Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg

120 125 130

CGT TCC CTG CCA GCA ACA CTA CCA CAG TGT CAG GCA GCG AAC AAG ACC 589

Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr

135 140 145

TGC CCC ACC AAT TAC ATG TGG AAT AAT CAC ATC TGC AGA TGC CTG GCT 637

Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala

150 155 160 165

CAG GAA GAT TTT ATG TTT TCC TCG GAT GCT GGA GAT GAC TCA ACA GAT 685

Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp

170 175 180

GGA TTC CAT GAC ATC TGT GGA CCA AAC AAG GAG CTG GAT GAA GAG ACC 733

Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr

185 190 195

TGT CAG TGT GTC TGC AGA GCG GGG CTT CGG CCT GCC AGC TGT GGA CCC 781

Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro

200 205 210

CAC AAA GAA CTA GAC AGA AAC TCA TGC CAG TGT GTC TGT AAA AAC AAA 829

His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys

215 220 225

CTC TTC CCC AGC CAA TGT GGG GCC AAC CGA GAA TTT GAT GAA AAC ACA 877

Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr

230 235 240 245

TGC CAG TGT GTA TGT AAA AGA ACC TGC CCC AGA AAT CAA CCC CTA AAT 925

Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn

250 255 260

CCT GGA AAA TGT GCC TGT GAA TGT ACA GAA AGT CCA CAG AAA TGC TTG 973

Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu

265 270 275

TTA AAA GGA AAG AAG TTC CAC CAC CAA ACA TGC AGC TGT TAC AGA CGG 1021

Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg

280 285 290

CCA TGT ACG AAC CGC CAG AAG GCT TGT GAG CCA GGA TTT TCA TAT AGT 1069

Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser

295 300 305

GAA GAA GTG TGT CGT TGT GTC CCT TCA TAT TGG CAA AGA CCA CAA ATG 1117

Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met

310 315 320 325

AGC TAAGATTGTA CTGTTTTCCA GTTCATCGAT TTTCTATTAT GGAAAACTGT 1170

Ser

GTTGCCACAG TAGAACTGTC TGTGAACAGA GAGACCCTTG TGGGTCCATG CTAACAAAGA 1230

CAAAAGTCTG TCTTTCCTGA ACCATGTGGA TAACTTTACA GAAATGGACT GGAGCTCATC 1290

TGCAAAAGGC CTCTTGTAAA GACTGGTTTT CTGCCAATGA CCAAACAGCC AAGATTTTCC 1350

TCTTGTGATT TCTTTAAAAG AATGACTATA TAATTTATTT CCACTAAAAA TATTGTTTCT 1410

GCATTCATTT TTATAGCAAC AACAATTGGT AAAACTCACT GTGATCAATA TTTTTATATC 1470

ATGCAAAATA TGTTTAAAAT AAAATGAAAA TTGTATTTAT AAAAAAAAAA AAAAAA 1526

<210> 4

<211> 350

<212> PRT

<213> Artificial Sequence

<220>

<223> the deduced amino acid sequence of a truncated, biologically active form

of VEGF-2

<400> 4

Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys Cys Gln Leu

-24 -20 -15 -10

Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asn Leu Asn Ser

-5 1 5

Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr Asn Thr Glu

10 15 20

Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln Cys Met Pro

25 30 35 40

Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val Ala Thr Asn

45 50 55

Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys Gly Gly Cys

60 65 70

Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr Ser Tyr Leu

75 80 85

Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln Gly Pro Lys

90 95 100

Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg Cys Met Ser

105 110 115 120

Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg Arg Ser Leu

125 130 135

Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr

140 145 150

Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp

155 160 165

Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His

170 175 180

Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys

185 190 195 200

Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu

205 210 215

Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu Phe Pro

220 225 230

Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys

235 240 245

Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn Pro Gly Lys

250 255 260

Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu Leu Lys Gly

265 270 275 280

Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg Pro Cys Thr

285 290 295

Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val

300 305 310

Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met Ser

315 320 325

<210> 5

<211> 196

<212> PRT

<213> Artificial Sequence

<220>

<223> the amino acid sequence of PDGFa

<400> 5

Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala 1 5 10 15

His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg

20 25 30

Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu

35 40 45

Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg

50 55 60

Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu

65 70 75 80

Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys

85 90 95

Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro

100 105 110

Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg

115 120 125

Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg

130 135 140

Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys

145 150 155 160

Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu

165 170 175

Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp

180 185 190

Thr Asp Val Arg

195

<210> 6

<211> 241

<212> PRT

<213> Artificial Sequence

<220>

<223> the amino acid sequence of PDGFb

<400> 6

Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg

1 5 10 15

Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met

20 25 30

Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu

35 40 45

His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met

50 55 60

Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg

65 70 75 80

Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu

85 90 95

Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp

100 105 110

Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln

115 120 125

Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr

130 135 140

Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg

145 150 155 160

Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu

165 170 175

Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser

180 185 190

Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val

195 200 205

Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg

210 215 220

Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly

225 230 235 240

Ala

<210> 7

<211> 232

<212> PRT

<213> Artificial Sequence

<220>

<223> the amino sequence of VEGF

<400> 7

Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu

1 5 10 15

Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly

20 25 30

Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln

35 40 45

Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu

50 55 60

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu

65 70 75 80

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro

85 90 95

Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His

100 105 110

Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys

115 120 125

Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val

130 135 140

Arg Gly Lys Gly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr

145 150 155 160

Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp

165 170 175

Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys

180 185 190

His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn

195 200 205

Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr

210 215 220

Cys Arg Cys Asp Lys Pro Arg Arg

225 230

<210> 8

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> the sequence having the signature for the PDGF / VEGF family

<400> 8

Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn

1 5 10

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> M13-2 reverse primer

<400> 9

atgcttccgg ctcgtatg 18

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> M13-2 forward primer

<400> 10

gggttttccc agtcacgac 19

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> VEGF primer F4

<400> 11

ccacatggtt caggaaagac a 21

<210> 12

<211> 50

<212> DNA

<213> Artificial Sequence

<220>

<223> PCR oligonucleotide 5 'primer sequence

<400> 12

tgtaatacga ctcactatag ggatcccgcc atggaggcca cggcttatgc 50

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> PCR oligonucleotide 3 'primer sequence

<400> 13

gatctctaga ttagctcatt tgtggtct 28

<210> 14

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> 5 'primer sequence containing a BamH1 site

followed by 18 nucleotides of VEGF-2 coding

sequence starting from the initiation codon

<400> 14

cgcggatcca tgactgtact ctaccca 27

<210> 15

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> 3 'sequence containing complementary sequences

to an XbaI site, HA tag, XhoI site and the last 15

nucleotides of VEGF-2 coding sequence

(not including the stop codon)

<400> 15

cgctctagat caagcgtagt ctgggacgtc gtatgggtac tcgaggctca tttgtggtct 60

<210> 16

<211> 3974

<212> DNA

<213> Artificial Sequence

<220>

<223> pHE4a expression vector sequence

<400> 16

ggtacctaag tgagtagggc gtccgatcga cggacgcctt ttttttgaat tcgtaatcat 60

ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 120

ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 180

cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 240

tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 300

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 360

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 420

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 540

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 600

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 660

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 720

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 840

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 900

gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 960

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 1020

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcgtcga 1140

caattcgcgc gcgaaggcga agcggcatgc atttacgttg acaccatcga atggtgcaaa 1200

acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg 1260

aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc 1320

cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg 1380

atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg 1440

ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg 1500

gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga 1560

agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg 1620

ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact 1680

aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc 1740

tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa 1800

atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg 1860

cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt 1920

gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg 1980

atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg 2040

ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt 2100

tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg 2160

gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc 2220

tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg 2280

ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 2340

gcgcaacgca attaatgtaa gttagcgcga attgtcgacc aaagcggcca tcgtgcctcc 2400

ccactcctgc agttcggggg catggatgcg cggatagccg ctgctggttt cctggatgcc 2460

gacggatttg cactgccggt agaactccgc gaggtcgtcc agcctcaggc agcagctgaa 2520

ccaactcgcg aggggatcga gcccggggtg ggcgaagaac tccagcatga gatccccgcg 2580

ctggaggatc atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa 2640

ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc 2700

gaaccccaga gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc 2760

gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc 2820

tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc 2880

cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag 2940

gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg 3000

aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga 3060

ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg 3120

caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc 3180

tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc 3240

cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg 3300

gccagccacg atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg 3360

gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag 3420

cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga 3480

gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga 3540

tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact 3600

ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg ttcgcttgct 3660

gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc tacctgcttt 3720

ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt catccggggt 3780

cagcaccgtt tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc 3840

cctgagtgct tgcggcagcg tgaagcttaa aaaactgcaa aaaatagttt gacttgtgag 3900

cggataacaa ttaagatgta cccaattgtg agcggataac aatttcacac attaaagagg 3960

agaaattaca tatg 3974

<210> 17

<211> 112

<212> DNA

<213> Artificial Sequence

<220>

<223> the nucleotide sequence of the regulatory elements of

the pHE4a promotor

<400> 17

aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg gataacaatt aagatgtacc 60

caattgtgag cggataacaa tttcacacat taaagaggag aaattacata tg 112

<210> 18

<211> 419

<212> PRT

<213> Artificial Sequence

<220>

<223> VEGF-2 T103-L215

<400> 18

Met His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala

1 5 10 15

Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe

20 25 30

Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala

35 40 45

Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser

50 55 60

Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met

65 70 75 80

Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln

85 90 95

Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala

100 105 110

His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys

115 120 125

Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe

130 135 140

Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr

145 150 155 160

Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr

165 170 175

Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu

180 185 190

Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser

195 200 205

Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile

210 215 220

Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn

225 230 235 240

Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys

245 250 255

Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser

260 265 270

Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu

275 280 285

Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys

290 295 300

Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys

305 310 315 320

Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu

325 330 335

Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro

340 345 350

Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys

355 360 365

Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr

370 375 380

Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser

385 390 395 400

Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro

405 410 415

Gln met ser

<210> 19

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> 5 'primer (Nde I / START and 18nt of coding sequence)

<400> 19

gcagcacata tgacagaaga gactataaaa 30

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> 3 'primer (Asp718, STOP, and 15nt of coding sequence)

<400> 20

gcagcaggta cctcacagtt tagacatgca 30

<210> 21

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> 3 'primer (Asp 718, STOP, and 15nt of coding sequence)

<400> 21

gcagcaggta cctcaacgtc taataatgga 30

<210> 22

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> the sequence containing the BamHI restriction enzyme

site followed by 1 spacer nt to stay in-frame with the

vector-supplied signal peptide and 17 nt of coding

sequence bases of VEGF-2 protein

<400> 22

gcagcaggat cccacagaag agactataaa 30

<210> 23

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> the sequence containing the XbaI restriction enzyme

site followed by a stop codon and 17 nucleotides

complementary to the 3 'coding sequence of VEGF-2

<400> 23

gcagcatcta gatcacagtt tagacatgca 30

<210> 24

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> the sequence containing the BamHI restriction enzyme site followe

d by 1 spacer nt to stay in-frame with the vector-supplied signal

peptide, and 26nt of coding sequence bases of VEGF-2 protein

<400> 24

gcagcaggat cccacagaag agactataaa atttgctgc 39

<210> 25

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> the sequence containing the XbaI restriction site followed

by a stop codon and 21 nucleotides complementary to the

3 'coding sequence of VEGF-2

<400> 25

gcagcatcta gatcaacgtc taataatgga atgaac 36

<210> 26

<211> 55

<212> DNA

<213> Artificial Sequence

<220>

<223> the 5 'primer containing a Klenow-filled BamHI site and

40 nt of VEGF-2 coding sequence starting from the

initiation codon

<400> 26

gatcgatcca tcatgcactc gctgggcttc ttctctgtgg cgtgttctct gctcg 55

<210> 27

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> the 3 'primer containing a BamHI site and 16nt of VEGF-2

coding sequence not including the stop codon

<400> 27

gcagggtacg gatcctagat tagctcattt gtggtcttt 39

<210> 28

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 5 'primer complementary to the

desired region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning of

VEGF-2 M1-M263 into the expression vector, pC4

<400> 28

gactggatcc gccaccatgc actcgctggg cttcttctc 39

<210> 29

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 3 'primer complementary to the

desired region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning of

VEGF-2 M1-M263 into the expression vector, pC4

<400> 29

gactggtacc ttatcacata aaatcttcct gagcc 35

<210> 30

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 5 'primer complementary to the

desired region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning of

VEGF-2-M1-D311 into the expression vector, pC4

<400> 30

gactggatcc gccaccatgc actcgctggg cttcttctc 39

<210> 31

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 3 'primer complementary to the

desired region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning of

VEGF-2-M1-D311 into the expression vector, pC4

<400> 31

gactggtacc ttatcagtct agttctttgt gggg 34

<210> 32

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 5 'primer complementary to the

desired region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning of

VEGF-2-M1-D311 into the expression vector, pC4

<400> 32

gactggatcc gccaccatgc actcgctggg cttcttctc 39

<210> 33

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 3 'primer complementary to the

desired region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning of

VEGF-2-M1-D311 into the expression vector, pC4

<400> 33

gactggtacc tcattactgt ggactttctg tacattc 37

<210> 34

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 5 'primer (Bam HI and 26 nt of

coding sequence) complementary to the desired

region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning

of VEGF-2 T103-L215 into pC4Sig

<400> 34

gcagcaggat ccacagaaga gactataaaa tttgctgc 38

<210> 35

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> the oligonucleotide 3 'primer (Xba I, STOP, and 15nt of

coding sequence) complementary to the desired

region of VEGF-2 to permit Polymerase Chain

Reaction directed amplification and sub-cloning

of VEGF-2 T103-L215 into pC4Sig

<400> 35

cgtcgttcta gatcacagtt tagacatgca tcggcag 37

Claims (26)

(a) the N-terminal deletion fragment set forth in Formula m-396 of SEQ ID NO: 2; (b) a C-terminal deletion fragment set forth in Formula -23-n of SEQ ID NO: 2; (c) the N-terminal and C-terminal deletion fragments set forth in Formula m-n of SEQ ID NO: 2 and (d) the C-terminal deletion fragment set forth in Formula + 9-n of SEQ ID NO: 2 An isolated polynucleotide comprising a nucleic acid sequence that is at least 95% identical to a polynucleotide encoding a polypeptide selected from the group consisting of: The isolated polynucleotide of claim 1, wherein the polypeptide comprises amino acid residues S-205 to S-396 of SEQ ID NO: 2 and / or amino acid residues F-9 to R-203 of SEQ ID NO: 2. A composition comprising the isolated polynucleotide of claim 1. The isolated polynucleotide of claim 1, encoding a biologically active fragment of VEGF-2. The isolated polynucleotide of claim 1, encoding a polypeptide that binds to an antibody against VEGF-2. The polynucleotide of claim 1, further comprising a polynucleotide encoding a heterologous polypeptide. A vector comprising the polynucleotide of claim 1. 8. The vector of claim 7, wherein the polynucleotide is operably linked with a heterologous regulatory sequence. A host cell comprising the vector of claim 7 or the polynucleotide of claim 1. (a) culturing the host cell of claim 9 under conditions suitable for producing the polypeptide, and (b) recovering the polypeptide from the cell culture of step (a). A polypeptide produced by the method of claim 10. (a) the N-terminal deletion fragment set forth in Formula m-396 of SEQ ID NO: 2; (b) a C-terminal deletion fragment set forth in Formula -23-n of SEQ ID NO: 2; (c) the N-terminal and C-terminal deletion fragments set forth in Formula m-n of SEQ ID NO: 2 and (d) the C-terminal deletion fragment set forth in Formula + 9-n of SEQ ID NO: 2 An isolated polypeptide comprising a polypeptide at least 95% identical to an amino acid sequence selected from the group consisting of: The isolated polypeptide of claim 12 comprising amino acid residues S-205 to S-396 of SEQ ID NO: 2 and / or amino acid residues F-9 to R-203 of SEQ ID NO: 2. A composition comprising the isolated polypeptide of claim 1. A composition comprising a first polypeptide fragment comprising amino acid residues S-205 to S-396 of SEQ ID NO: 2 and a second polypeptide fragment comprising amino acid residues F-9 to R-203 of SEQ ID NO: 2. The isolated polypeptide of claim 12, which is a biologically active fragment. The isolated polypeptide of claim 12, which is antigenic. The isolated polypeptide of claim 12 further comprising a heterologous polypeptide. An antibody against the polypeptide of claim 12. A compound that activates the polypeptide of claim 12. A compound that inhibits the polypeptide of claim 12. Preventing a medical disease comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of claims 12 and 13, a composition of claims 3, 14 and 15, or a polynucleotide of claims 1 and 2. , How to treat or alleviate. (a) measuring the presence or absence of a mutation in the polynucleotide of claim 1, and (b) diagnosing susceptibility to a pathological disease or pathological condition based on the presence or absence of such mutations, including pathological disease or pathology in a subject associated with the expression or activity of a particular secreted protein. How to diagnose susceptibility to enemy disease. (a) measuring the presence or expression amount of the polypeptide of claim 12 in a biological sample, and (b) diagnosing susceptibility to a pathological disease or pathological condition based on the presence or amount of expression of such polypeptides, the pathological disease or pathology in a subject associated with the expression or activity of a particular secreted protein How to diagnose susceptibility to enemy disease. (a) contacting the polypeptide of claim 12 with a binding partner, and (b) determining whether the binding partner exhibits the activity of the polypeptide, wherein the binding partner for the polypeptide of claim 10 is identified. (a) expressing the polypeptide of claim 12 in a particular cell; (b) separating the supernatant; (c) detecting activity in the biological assay, and (d) identifying the protein of interest in a supernatant having such activity Comprising, a method for identifying a specific activity in a biological assay.