MXPA00008920A - Vascular endothelial growth factor 2 - Google Patents

Abstract

Disclosed are human VEGF-2 polypeptides, biologically active, diagnostically or therapeutically useful fragments, analogs, or derivatives thereof, and DNA(RNA) encoding such VEGF-2 polypeptides. Also provided are procedures for producing such polypeptides by recombinant techniques and antibodies and antagonists against such polypeptides. Such polypeptides and polynucleotides may be used therapeutically for stimulating wound healing and for vascular tissue repair. Also provided are methods of using the antibodies and antagonists to inhibit tumor angiogenesis and thus tumor growth, inflammation, diabetic retinopathy, rheumatoid arthritis, and psoriasis.

Description

VASCULAR E3ÜDOTELIAL GROWTH FACTOR 2 BACKGROUND OF THE INVENTION The present invention relates to newly identified polynucleotides, polypeptides encoded by these polynucleotides, the use of these polynucleotides and polypeptides, as well as the production of these polynucleotides and polypeptides. The polypeptides of the present invention have been identified as members of the vascular endothelial growth factor family. More particularly, the polypeptides of the present invention are the vascular endothelial growth factor of human 2 (VEGF-2). The invention also relates to the inhibition of the action of these polypeptides. The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth and tissue repair. Angiogenesis is also an essential part of certain pathological conditions, such as neoplasia. { that is, tumors and gliomas). Abnormal angiogenesis is associated with other diseases such as inflammation, rheumatoid arthritis, psoriasis and diabetic retinopathy REF: 122514 (Folk an, J. and Klagsbrun, M., Science 235: 442-441 (1987)). Fibroblast growth factor molecules, both acidic and basic, are indolent for endothelial cells and other cell types. Angiotropin and angiogenin may induce angiogenesis, although their functions are unclear (Folkman, J., Cancer Medi cine, Lea and Febiger Press, pages 153-170 (1993)). A highly selective mitogen for endothelial, vascular cells is the endothelial, vascular or VEGF growth factor (Ferrara, N. et al., Endocr. Rev. 23: 19-32 (1992)), which is also known as a factor of vascular permeability (VPF, for its acronym in English). Vascular endothelial growth factor is an angiogenic, secreted mitogen whose target cell specificity seems to be restricted to endothelial, vascular cells. The murine VEGF gene has been characterized and its pattern of expression in embryogenesis has been analyzed. A persistent expression of VEGF was observed in the epithelial cells adjacent to the fenestrated endothelium, for example, in the choroid plexus and kidney glomeruli. The data were consistent with a role of VEGF as a multifunctional regulator of the growth and differentiation of endothelial cells (Breier, G. et al, Devel opmen t 114: 521-532 (1992)). VEGF shares sequence homology with growth factors derived from human platelets, PDGFa and PDGFb (Leung, D.., Et al., Sci en 246: 1306-1309, (1989)). The degree of homology is approximately 21% and 23%, respectively. Eight cysteine residues that contibute to the disulfide bond formation are strictly conserved in these proteins. Although these are similar, there are specific differences between VEGF and PDGF. While PDGF is a major growth factor for connective tissue, VEGF is highly specific for endothelial cells. Alternatively, the bound mRNAs have been identified for both VEGF, PLGF and PDGF and these different binding products differ in biological activity and receptor binding specificity. VEGF and PDGF function as homo-dimers or hetero-dimers and bind to the receptors which produce the intrinsic tyrosine kinase activity after dimerization of the receptor. VEGF has four different amino acid forms 121, 165, 189 and 206 due to the alternative binding. VEGF121 and VEGF165 are soluble and are capable of promoting angiogenesis, while VEGF189 and VEGF-206 bind to proteoglycans containing heparin on the surface of cells. The temporal and spatial expression of VEGF has been correlated with the physiological proliferation of blood vessels (Gajdusek, CM, and Carbón, SJ, Cel l Physiol., 139: 510-519 (1989); McNeil, PL, et al., J. Cell Biol. 205: 811-822 (1989)). Their high affinity binding sites are located only in the endothelial cells in the tissue sections (Jakeman, L.B., and collaborators, Cl in. Inves t 89: 24 4 - 253 (1989)). The factor can be isolated from pituitary cells and various lines of tumor cells, and has been implicated in some human gliomas (Piet, K.H., Na ture 359: 845-848 (1992)). Interestingly, the expression of VEGF121 or VEGF165 confers in xylens of Chinese hamster ovary the ability to form tumors in nude mice (Ferrara, N. et al, J. Clin.Inves.t.91: 160-110 (1993)) . It is shown that inhibition of VEGF function by anti-VEGF monoclonal antibodies inhibits tumor growth in immuno-deficient mice (Kim, K.J., Na ture 362: 841- 844 (1993)). In addition, it has been shown that a dominant-negative mutation of the VEGF receptor inhibits the growth of glioblastomas in mice.

It has also been found that the vascular permeability factor (VPF) is responsible for the persistent microvascular hyperpermeability of plasma proteins even after the cessation of a lesion, which is a characteristic feature of the healing of normal wounds. This suggests that VPF is an important factor in wound healing. Brown, L.F. and collaborators, J. Exp. Med. 1 76: 1315-131 9 (1992). The expression of VEGF is high in vascularized tissues,. { for example, lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. It has also been shown that VEGF induces angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissues, especially vascular tissues, VEGF 'has been proposed for use in the promotion of vascular tissue repair (for example, in atherosclerosis). US Patent No. 5,073,492, issued December 17, 1991 to Chen et al., Describes a method for synergistically increasing the growth of endothelial cells in an appropriate environment, which comprises adding to the environment, VEGF, effectors and a derivative factor. of the serum. Also, the DNA of the C-subunit of vascular endothelial cell growth factor has been prepared by polymerase chain reaction techniques. DNA encodes a protein that can exist as either a heterodimer or a homodimer. The protein is a mitogen of endothelial, vascular, mammalian cells and, as such, is useful for the promotion of vascular development and repair, as described in European patent application No. 92302750.2, published September 30, 1992. .

Brief Description of 2nd Invention The polypeptides of the present invention have been putatively identified as a novel vascular endothelial growth factor based on the homology of amino acid sequences to human VEGF. In accordance with one aspect of the present invention, new fully developed or mature polypeptides are provided, as well as diagnostically or therapeutically useful fragments, analogs and biologically active derivatives thereof. The polypeptides of the present invention are of human origin. According to another aspect of the present invention, isolated nucleic acid molecules comprising polynucleotides encoding the full-length or truncated VEGF-2 polypeptides having amino acid sequences shown in SEQ ID NOS: 2 or 4 are provided. respectively, or the amino acid sequences encoded by the cDNA clones deposited in bacterial hosts such as ATCC Repository Number 97149 on May 12, 1995 or ATCC Repository Number 75698 on March 4, 1994. The present invention also refers to fragments, analogs and biologically active and diagnostically or therapeutically useful derivatives of VEGF-2. In accordance with yet another aspect of the present invention, processes for the production of such polypeptides are provided by recombinant techniques comprising culturing the prokaryotic and / or eukaryotic host cells, recombinants, which contain a nucleic acid sequence encoding a polypeptide of the present invention, under conditions that promote the expression of proteins and the subsequent recovery of proteins. In accordance with yet a further aspect of the present invention, processes are provided for using such polypeptides, or polynucleotides that encode such polypeptides for therapeutic purposes, for example, to stimulate angiogenesis, wound healing, bone and tissue growth. damaged, and to promote vascular tissue repair. In particular, processes for using such polypeptides or polynucleotides encoding such polypeptides are provided for the treatment of a peripheral artery disease, such as ischemia of a critical limb and coronary artery disease. In accordance with yet another aspect of the present invention, antibodies against such polypeptides and processes for producing such polypeptides are provided. In accordance with yet another aspect of the present invention, antagonists to such polypeptides are provided, which can be used to inhibit the action of such polypeptides, for example, to prevent tumor angiogenesis and thus inhibit growth. of tumors, to treat diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis. In accordance with another aspect of the present invention, nucleic acid test substances are provided which comprise nucleic acid molecules of sufficient length to specifically hybridize to the nucleic acid sequences of the present invention. In accordance with another aspect of the present invention, methods are provided for diagnosing diseases or susceptibility to diseases related to mutations in the nucleic acid sequences of the present invention and proteins encoded by these nucleic acid sequences. According to yet another additional aspect of the present invention, there is provided a process for using such polypeptides, or polynucleotides encoding such polypeptides, for purposes related to scientific research, DNA synthesis and the preparation of DNA vectors. . These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are illustrative of the embodiments of the invention and are not intended to limit the scope of the invention encompassed by the claims. Figures 1A-1E show the full-length nucleotide (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) of VEGF-2. The polypeptide comprises approximately 419 amino acid residues of which approximately 23 represent the leader sequence. One-letter abbreviations are used, normal for amino acids. Sequencing was performed using the Model 373 Automated DNA Sequencer (Applied Biosystems, Inc.). The sequence ordering accuracy is predicted to be greater than 97%. Figures 2A-2D show the nucleotide (SEC ID NO: 3) and the deduced amino acid sequence (SEQ ID NO: 4) of a truncated, biologically active form of VEGF-2. The polypeptide comprises about 350 amino acid residues of which approximately the first 24 amino acids represent a leader sequence. Figures 3A-3B are an illustration of the homology of amino acid sequences between PDGFa (SEQ ID NO: 5), PDGFb (SEQ ID NO: 6) VEGF (SEQ ID N0f7), and VEGF-2 (SEQ. ID NO: 4). The areas in the tables indicate the conserved sequences and the location of the eight conserved cysteine residues. Figure 4 shows, in table form, the percentage of homology between PDGFa, PDGFb, VEGF and VEGF-2.

Figure 5 shows the presence of VEGF-2 mRNA in human breast tumor cell lines. Figure 6 depicts the results of a Northern blot analysis of VEGF-2 in human adult tissues. Figure 7 shows a photograph of an SDS-PAGE gel after in vitro transcription, translation and electrophoresis of the polypeptide of the present invention. Band 1: marker 14C and RAINBO M.W .; Band 2: FGF control; Band 3: VEGF-2 produced by M13-reverse and forward primers; Band 4: VEGF-2 produced by primers of M13-inverse and VEGF-F4; Band 5: VEGF-2 produced by primers of M13-inverse and VEFG-F5. Figures 8A and 8B depict photographs of SDS-PAGE gels. The VEGF-2 polypeptide was expressed in a baculovirus system consisting of Sf9 cells. The medium protein and the cytoplasm of the cells were analyzed by SOS-PAGE under non-reduced conditions (Figure 8A) and reduced (Figure 8B). Figure 9 depicts a photograph of an SDS-PAGE gel. The medium of the Sf9 cells "infected with a nucleic acid sequence of the present invention was precipitated." The precipitated, resuspended product was analyzed by SDS-PAGE and stained with Coomassie brilliant blue Figure 10 depicts a photograph of a gel of SDS-PAGE The VEGF-2 was purified from the supernatant of the medium and analyzed by SDS-PAGE in the presence or absence of the b-mercaptoethanol reduction agent and stained by Coomassie brilliant blue. Inverse phase CLAP of purified VEGF-2 using a RP-300 column (0.21 x 3 cm, Applied Biosystems, Inc.) The column was equilibrated with 0.1% trifluoroacetic acid (Solvent A) and the proteins eluted with a 7.5 gradient. Min from 0 to 60% of Solvent B, composed of 0.07% TFA containing acetonitrile, * elution of proteins was monitored by absorbance at 215 nm ("red" line) and 280 nm ("blue" line). of Solvent B is shown by the "green" line Figure 12 is a bar graph illustrating the effect of partially purified VEGF-2 protein on vascular endothelial cell growth compared to basic fibroblast growth factor. Figure 13 is a bar graph illustrating the effect of purified VEGF-2 protein on vascular endothelial cell growth. Figure 14 depicts the expression of VEGF-2 mRNA in fetal and adult human tissues. Figure 15 depicts the expression of VEGF-2 mRNA in human primary culture cells. Figure 16 represents the transient expression of the VEGF-2 protein in COS-7 cells. Figure 17 depicts the proliferation stimulated by VEGF-2 of endothelial cells of the human umbilical vein (HUVEC, for its acronym in English ) . Figure 18 depicts the proliferation stimulated by VEGF-2 of icrovascular, dermal endothelial cells. Figure 19 depicts the stimulatory effect of VEGF-2 on the proliferation of microvascular endothelial, umbilical cord, endometrial and aortic bovine cells. Figure 20 depicts the inhibition of proliferation of smooth muscle cells (human aortic), vascular induced by PDGF. Figure 21 depicts the stimulation of the migration of HUVEC and bovine microvascular endothelial cells (BMEC) by VEGF-2.

Figure 22 depicts the stimulation of nitric oxide release from HUVEC by VEGF-2 and VEGF-1. Figure 23 depicts the inhibition of microvascular endothelial cell (CADMEC) cord formation by VEGF-2. Figure 24 depicts the stimulation of angiogenesis by VEGF, VEGF-2 and bFGF in the CAM assay. - Figure 25 represents the restoration of certain parameters in the ischemic limb by the VEGF-2 protein (Figure 25, upper panels) and the pure expression plasmid (Figure 25, intermediate panels): PS ratio (Figure 25a ") Blood Flow and Flow Reserve (Figure 25b), Angiographic Record (Figure 25c), Capillary Density (Figure 25d) Figures 26 AG represent the ability of VEGF-2 to affect blood pressure, diastolic in spontaneously hypertensive rats (SHR , Figures 26a and b represent the dose-dependent decrease in blood pressure, diastolic achieved with VEGF-2 (Figures 26c and d represent average, lowered arterial pressure (MAP, for short in English) observed with VEGF-2, Panel E shows the effect of increasing dose of VEGF-2 on average blood pressure (MAP) of SHR rats, Panel F shows the effect of VEGF-2 on diastolic pressure. of SHR rats Panel G shows the effect of VEGF-2 on blood pressure, diastolic of SHR rats. Figure 27 represents the inhibition of VEGF-2N = and the proliferation induced by VEGF-2. Figure 28 shows a schematic representation of the expression vector of pHE4a (SEQ ID NO: 16). The locations of the kanamycin resistance marker gene, the linker region of the multiple cloning site, the oriC sequence and the laclq coding sequence are indicated. Figure 29 shows the nucleotide sequence of the regulatory elements of the pHE4a promoter (SEQ ID NO: 17). The two sequences are indicated c, the Shine-Delgarno sequence (S / D) and the terminal restriction sites HindI I? and Ndel (in italics.

Detailed Description of Preferred Modalities In accordance with one aspect of the present invention, there are provided isolated nucleic acid molecules comprising a polynucleotide encoding a VEGF-2 polypeptide having the deduced amino acid sequence of Figure 1 (SEQ. ID N0: 2), which was determined by sequencing a cloned cDNA. The nucleotide sequence shown in SEQ ID NO: 1 was obtained by sequencing a cDNA clone, which was deposited on May 12, 1995 at the American Type Tissue Collection (ATCC), 10801 University Boulevard, Manassas , VA 20110-2209, and was given ATCC Repository No. 97149. In accordance with another aspect of the present invention, isolated nucleic acid molecules are provided which comprise a polynucleotide encoding a truncated VEGF-2 polypeptide that has the deduced amino acid sequence of Figure 2 (SEQ ID NO: 4), which was determined by sequencing a cloned cDNA. The nucleotide sequence shown in SEQ ID NO: 3 was obtained by sequencing a cDNA clone, which was deposited on March 4, 1994 at the American Type Tissue Collection (ATCC), 10801 University Boulevard, Manassas , VA 20110-2209, and was given the ATCC Deposit Number 75698. Unless otherwise indicated, all nucleotide sequences determined by ordering the sequences of a DNA molecule herein were determined using an automated DNA sequencer (such as Model 373 from Applied Biosystems, Inc.) and all amino acid sequences of polypeptides encoded by the DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art, for any DNA sequence determined by this automatic approach, any nucleotide sequence determined in the present may contain some errors. The nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequentially ordered DNA molecule. The actual sequence can be determined more precisely by other approaches including manual methods of ordering DNA sequences well known in the art. As is also known in the art, an individual insertion or deletion in a given nucleotide sequence compared to the actual sequence will cause a change in the translation structure of the. nucleotide sequence such that the predicted amino acid sequence, encoded by a given nucleotide sequence, sexes completely different from the amino acid sequence actually encoded by the ordered DNA molecule in sequence, starting at the point of this insertion or deletion. A polynucleotide encoding a polypeptide of the present invention can be obtained from human embryo osteoclastomas in the first stage (week 8 to 9), adult heart or several breast cancer cell lines. The polynucleotide of this invention was discovered in a cDNA library derived from the human embryo in the first stage, of 9 weeks. It is structurally related to the VEGF / PDFG family. It contains an open reading frame that encodes a protein of approximately 419 amino acid residues of which approximately the first 23 amino acid residues are the putative leader sequence such that the fully developed protein comprises 396 amino acids and the protein exhibiting the homology of higher amino acid sequences to human vascular endothelial growth factor (30% identity), followed by PDGFa (24%) and PDGFb (22%). (See Figure 4). It is especially important that all eight cysteines be conserved within the four family members (see the areas in the tables in Figure 3). In addition, the identification for the PDGF / VEGF family, PXCVXXXRCXGCCN, (SEQ ID NO: 8) is conserved in VEGF-2 (see Figure 3). The homology between VEGF-2, VEGF and the two PDGFs is at the level of protein sequences. No homology of nucleotide sequences can be detected, and therefore, it would be difficult to isolate VEGF-2 through simple approaches such as low accuracy or austerity hybridization. The VEGF-2 polypeptide of the present invention is proposed to include the full length polypeptide and polynucleotide sequence which is encoded by any leader sequence and by active fragments of the full length polypeptide. The active fragments are understood to include portions of the full-length amino acid sequence which has less than 419 complete amino acids of the full length amino acid sequence as shown in SEQ ID NO: 2, but still contain the eight cysteine residues shown, conserved in Figure 3 and still having VEGF-2 activity. There are at least two alternatively linked VEGF-2 mRNA sequences present in normal tissues. The two bands in Figure 7, band 5 indicates the presence of the alternatively linked mRNA encoding the VEGF-2 polypeptide of the present invention. The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, the DNA including cDNA, genomic DNA and synthetic DNA. The DNA can be double-stranded or single-stranded, and if it is single-stranded, it can be the coding chain or non-coding (anti-sense) chain. The coding sequence which encodes the fully developed polypeptide may be identical to the coding sequence shown in Figure 1 or Figure 2, or that of the deposited clones, or it may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same, fully developed polypeptide as the DNA of Figure 1, Figure 2, or the deposited cDNAs. - The polynucleotide which encodes the fully developed polypeptide of Figure 1 or Figure 2 or fully developed polypeptides, encoded by the deposited cDNAs may include: only the coding sequence for the fully developed polypeptide; the coding sequence for the fully developed polypeptide and the additional coding sequences such as a leader or secretory sequence or a sequence of proproteins; the coding sequence for the fully developed polypeptide (and the optionally additional coding sequences) and the non-coding sequences, such as the introns or the 5 'and / or 3' non-coding sequence of the coding sequence for the fully developed polypeptide. In this manner, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only the coding sequences for the polypeptide as well as a polynucleotide which includes the additional coding and / or non-coding sequences. The present invention further relates to variants of the polynucleotides described hereinbefore which encode fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figures 1 or 2, or the polypeptide encoded by the cDNA of the clones. deposited. The variant of the polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides that encode the same fully developed polypeptide as shown in Figures 1 or 2 or the fully developed polypeptide itself, encoded by the cDNA of the deposited clones as well as variants of such polynucleotides, variants encoding a fragment, a derivative or an analogue of the polypeptides of Figures 1 or 2, or the polypeptide encoded by the cDNA of the deposited clones. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants. As indicated hereinabove, the polynucleotide may have a coding sequence which is an allelic variant that occurs naturally from the coding sequence shown in Figures 1 or 2, or from the coding sequence of the deposited clones. As is known in the art, an allelic variant is an alternate form of a polynucleotide sequence which has a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide. The present invention also includes polynucleotides, wherein the coding sequence for the fully developed polypeptide can be fused in the same reading frame to a polynucleotide which aids in the expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence to control the transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence divided by the host cell to develop the mature form of the polypeptide. The polynucleotides can also be encoded by a proprotein which is the fully developed protein plus the additional 5 'amino acid residues. A fully developed protein that has a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is divided, a fully developed, active protein remains. Thus, for example, the polynucleotide of the present invention can be encoded by a fully developed protein, or by a protein having a prosequence or by a protein having both a prosequence and a pre-sequence (leader sequence).

The polynucleotides of the present invention may also have the coding sequence fused in the structure to a marker sequence which allows purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the fully developed polypeptide fused to the tag in the case of a bacterial host, or, for example, the tag sequence may be a tag of haemagglutinin (HA) when a mammalian host is used, for example COS-7 cells. The HA tag corresponds to an epitope derived from the influenza hemaglutanin protein (ilson, I., et al., Cell 37: 161 (1984)). Additional embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having at least 95% identical nucleotide sequence, and most preferably at least 96%, 97%, 98% or 99% identical to ( a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO: 2; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO: 2, but lacking N-terminal methionine; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 1 to about 396 in SEQ ID NO: 2; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Repository No. 97149; (e) a nucleotide sequence encoding the fully developed VEGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Repository No. 97149; or (f) a complementary nucleotide sequence for any of the nucleotide sequences in (a), (b), (c), (d) or (e). In addition, embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 95% identical, and more preferably at least 96%, 97%, 98% or 9"9"% identical to (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO: 4; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence in SEQ ID NO: 4, but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the polypeptide having the amino acid sequence at positions from about 1 to about 326 in SEQ ID NO: 4; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Repository No. 75698; (e) a nucleotide sequence encoding the fully developed VEGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75698; or (f) a complementary nucleotide sequence for any of the nucleotide sequences in (a), (b), (c), (d) or (e). By a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a VEGF-2 polypeptide, it is proposed that the nucleotide sequence of the polynucleotide is identical to the sequence of reference except that the polynucleotide sequence may include up to five dot mutations per 100 nucleotides of the reference nucleotide sequence encoding the VEGF-2 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the complete nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5N or 3N terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually between the nucleotides in the reference sequence or in one or more groups contiguous within the reference sequence. As a practical matter, if any particular nucleic acid molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide sequence shown in SEQ ID NOS: 1 or 3 , or to the nucleotide sequence of the deposited cDNA clone (s), can be determined conventionally 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, Wl 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Ma thema ti cs 2: 482-489 (1981), to find the best homology segment between two sequences. When Bestfit or any other sequence alignment program is used to determine if a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are adjusted, of course, such that the percentage of Identity is calculated on the full length of the reference nucleotide sequence and that openings or ranges in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. As described in detail below, the polypeptides of the present invention can be used to elevate the polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting the expression of the VEGF-2 protein as described below or as agonists and antagonists capable of increasing or inhibiting the function of the VEGF-2 protein. In addition, such polypeptides can be used in the hybrid system of two yeasts to "capture" proteins that bind to the VEGF-2 protein which are also candidate agonists and antagonists according to the present invention. The hybrid system of two yeasts is described in Fields and Song. nature 340: 245-246 (1989).

In another aspect, the invention provides a peptide or a polypeptide comprising a portion carrying an epitope of a polypeptide of the invention. As for the selection of peptides or polypeptides that "carry an antigenic epitope (i.e., which contains a region of a protein molecule to which an antibody can bind), it is well known in the art that synthetic peptides , relatively short that mimic part of a protein sequence are usually capable of producing an antiserum that reacts with the partially imitated protein, see, for example, Sutcliffe, JG, Shinnick, TM, Green, N. and Learner, RA (1983). Antibodies that react with predetermined protein sites Science 219: 660-666 Peptides capable of producing protein-reactive sera are often represented in the primary sequence of a protein, can be characterized by a set of rules chemical, simple, and are not confined to immunodominant regions of intact proteins (ie, immunogenic epitopes) or amino or carboxyl termini. eptides that are extremely hydrophobic and those of six or less residues are generally not effective in inducing antibodies that bind to the mimicked protein; Soluble, larger peptides, especially those containing proline residues, are usually effective. Sutcliffe et al., Supra, at 661. For example, 18 to 20 peptides designated according to these guidelines, containing 8-39 residues covering 75% of the polypeptide chain sequence of HAI haemagglutinin from influenza virus, induced antibodies that react with the HAI protein or an intact virus; and 12/12 peptides of MuLV polymerase and 18/18 of the antibodies induced by the rabies glycoprotein that precipitate the respective proteins. Therefore, the peptides and polypeptides carrying the antigenic epitope of the invention are useful for raising antibodies, including monoclonal antibodies, that specifically bind to a polypeptide of the invention. In this way, a high proportion of hybridomas obtained by the fusion of cells of the donor vessel immunized with a peptide carrying the antigenic epitope generally secrete antibodies reactive with the native protein. Sutcliffe et al., Supra, at 663. Antibodies raised by the peptides or polypeptides bearing the antigenic epitope are useful for detecting the imitated protein, and antibodies for different peptides can be used to locate the fate of various regions of a precursor. the protein which is subjected to post-translational processing. Peptides and anti-peptide antibodies can be used in a variety of qualitative or quantitative assays for the mimicked protein, for example in competition assays since it has been shown that even short peptides (eg, 9 amino acids) can bind and displace the larger peptides in the immunoprecipitation assays. See, for example, Wilson et al., Cell 37: 767-778 (1984) at 777. The anti-peptide antibodies of the invention are also useful for the purification of the protein mimicked, for example, by adsorption chromatography using methods well known in the art. Peptides and polypeptides bearing antigenic epitopes of the invention designated according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and much more preferably between about 15 to about 30 amino acids contained within of the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30, 40, 50, 60, 70, 80, 90, 100 or 150 amino acids, or any length up to and including the complete amino acid sequence of a polypeptide of the invention, are also considered epitope-bearing peptides or polypeptides of the invention and are also useful for inducing the antibodies that they react with the imitated protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (ie, the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred. Non-limiting examples of the antigenic polypeptides or peptides that can be used to generate antibodies specific for VEGF-2 include the following: a polypeptide comprising amino acid residues from about leu-37 to about glu-45 in SEQ ID NO: 2, from about Tyr-58 to about Gly-66 at SEQ ID NO: 2, from about Gln-73 to about Glu-81 at SEQ ID NO: 2, from about Asp-100 to about Cys-108 at SEQ ID NO: 2, from about Gly-140 to about Leu-148 at SEQ ID NO: 2, from about Pro-168 to about Val-176 at SEQ ID NO: 2, from about His-138 to about Lys -191 in the SEQ ID NO: 2, from about Ile-201 to about Thr-209 in SEQ ID NO: 2, from about Ala-216 to about Tyr-224 in the SEQ ID NO: 2, from about Asp-244 to about His-254 at SEQ ID NO: 2, from about Gly-258 to about Glu-266 at SEQ ID NO: 2, from about Cys-272 to about -280 in SEQ ID NO: 2, from approximately Pro-283 to approximately Ser-291 in the SEQ ID NO: 2, from about Cys-296 to about Gln-304 in SEQ ID NO: 2, from about Ala-307 to about Cys-316 in the SEQ ID NO: 2, from about Val-319 to about Cys-335 at SEQ ID NO: 2, from about Cys-339 to about Leu-347 at SEQ ID NO: 2, from about Cys-360 to about Glu-373 in SEQ ID NO: 2, from about Tyr-378 to about Val-386 in the SEQ ID NO: 2, and from about Ser-388 to about Ser-396 in SEQ ID NO: 2. It has been determined that these polypeptide fragments carry the antigenic epitopes of the VEGF-2 protein by analyzing the antigenic index of Jameson- Wolf. The peptides and epitope-bearing polypeptides of the invention can be produced by any conventional means for making peptides or polypeptides including recombinant means using the nucleic acid molecules of the invention. For example, a short epitope-bearing amino acid sequence can be fused to a larger polypeptide that acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. Peptides carrying epitopes can also be synthesized using known methods of chemical synthesis. For example, Houghten has described a simple method for the synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing individual amino acid variants of a segment of the HAI polypeptide which are prepared and they are characterized (by binding studies of the ELISA type) in less than four weeks. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides; specificity of antigen-antobody interaction at the level of individual amino acids.

Proc. Ma tl. Acad. Sci. USA 82: 5131-5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,211 issued to Houghten et al. (1986). In this procedure, the individual resins for the solid phase synthesis of various peptides are contained in separate solvent-permeable packages, which make possible the optimal use of the many repetitive, identical steps involved in IOJS solid-phase methods. A completely manual procedure allows 500-1000 or more synthesis to be conducted simultaneously. Houghten et al., Supra, at 5134. The peptides and polypeptides bearing epitopes of the invention are used to induce the antibodies according to methods well known in the art. See, for example, Sutcliffe et al., Supra; Wilson et al., Supra; Chow, M. et al., Proc. Na ti. Acad. Sci. USA 82: 910-914; and Bittle, F. J. et al., J. Gen. Virol. 66: 2341-2354 (1985). In general, animals can be immunized with the free peptide; however, the anti-peptide antibody titer may be increased by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. 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 can be coupled to a carrier using a more general binding agent such as glutaraldehyde . Animals such as rabbits, rats and mice are immunized with either free peptides or coupled to the carrier, for example, by intraperitoneal and / or intradermal injection of emulsions containing approximately 100 mg of peptide or carrier protein and Freund's adjuvant. Several booster injections may be necessary, for example, at intervals of about two weeks, to provide a useful titre of anti-peptide antibody which can be detected, for example, by ELISA assay using a free peptide adsorbed to a surface solid The titre of anti-peptide antibodies in the serum of an immunized animal can be increased by the selection of the anti-peptide antibodies, for example, by adsorption to the peptide on a solid support and elution of the antibodies selected according to the methods well known in the art. Peptides carrying immunogenic epitopes of the invention, ie, those portions of a protein that produce an antibody response when the entire protein is an immunogenic substance, are identified according to methods known in the art. For example, Geysen et al., Supra, describe a procedure for the concurrent, rapid synthesis in solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. The interaction of the peptides synthesized with the antibodies is then easily detected without separating them from the support. In this way, a peptide carrying an immunogenic epitope of a desired protein can usually be identified by one of ordinary skill in the art. For example, the inumunologically important epitope in the coat protein of the foot and mouth disease virus was localized by Geysen et al. With a resolution of seven amino acids by synthesizing a set of superposition of all 208 possible hexapeptides that cover the sequence Complete of 213 amino acids of the protein. Then, a complete, complete set of peptides in which all 20 amino acids were substituted in turn were synthesized at each position within the epitope, and the particular amino acids conferring specificity for the reaction with the antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention can usually be made by this method. U.S. Patent No. 4,708,781 issued to Geysen (1987) further describes this method of identifying a peptide carrying an immunogenic epitope of a desired protein. Still further, U.S. Patent No. 5,194,392 issued to Geysen (1990) discloses a general method for detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (ie, an Amimotope) on the which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 issued to Geysen (1989) discloses a method for detecting or determining a monomer sequence which is a topographic equivalent of a ligand which is complementary to the ligand binding site of a particular receptor. of interest. Similarly, the North American patent No. 5,480,971 issued to Houghten, R. A. et al. (1996) in Peralkylated Oligopeptide Mixtures describes peralkylated oligopeptides with alkyl of 1 to 7 carbon atoms, linear and assemblies and libraries of such peptides, as well as methods for using such sets of oligopeptides and libraries to determine the sequence of a prealkylated oligopeptide which binds preferentially to an acceptor molecule of interest. Thus, the non-peptide analogs of the epitope-bearing peptides of the invention can also be customarily made by these methods. As one of skill in the art will appreciate, the VEGF-2 polypeptides of the present invention and epitope-bearing fragments thereof, described above, can be recombined with portions of the constant domain of immunoglobulins.

(IgG), resulting in chimeric polypeptides.

These fusion proteins facilitate purification and show an increased life period. This has been shown, for example, for chimeric proteins consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy and light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Na t ure 331: 84-8 (1988) In accordance with the present invention, novel variants of VEGF-2 are also described, which can be produced by removing or substituting one or more amino acids of VEGF-2. they are called allelic variations, the allelic variations can be inactive (they do not change in the encoded polypeptide) or they can have an altered amino acid sequence In order to try to improve or alter the characteristics of the native VEGF-2, the The recombinant DNA technology, known to those skilled in the art, can be used to create new polypeptides, muteins and deletions. they can show, for example, increased activity or increased stability. In addition, these could be purified at a higher yield and could show better solubility at least under certain purification and storage conditions. Examples of mutations that can be constructed are subsequently exposed.

Deletions of telagrafico and carboxy terminal amino In addition, VEGF-2 appears to be proteolytically divided into the expression that results in polypeptide fragments of the following sizes when passed on an SDS-PAGE gel (sizes are approximate) (See, Figures 6-8, for example): 80, 59, 45, 41, 40, 39, 38, 37, 36, 31, 29, 21 and 15 kDa. These polypeptide fragments are the result of the proteolytic cleavage - in both the N-terminal and C-terminal portions of the protein. These proteolytically generated fragments appear to have activity, particularly the 21 kDa fragment. In addition, the design of proteins can be employed in order to improve or alter one or more characteristics of native VEGF-2. The suppression of carboxy-terminal amino acids can increase the activity of proteins. An example is gamma interferon which shows up to ten times higher activity by removing ten amino acid residues from the carboxy terminus of the protein (Dóbeli et al., J. of Biotechnology 7: 199-216 (1988)). Thus, one aspect of the invention is to provide the polypeptide analogs of VEGF-2 and the nucleotide sequences encoding such analogs that exhibit enhanced stability (eg, when exposed to pH, thermal, typical or other environmental conditions). storage) relative to the native VEGF-2 polypeptide. Particularly preferred VEGF-2 polypeptides are shown below (numbering starts with the first amino acid in the protein (Met) (Figure 1 (SEQ ID NO: 18): Ala (residue 24) to be (residue 419); Pro (25) to Ser (419); Wing (26) to Ser (419); Wing (27) to Ser (419); Ala (28) to Ser (419); Wing (29) to Ser (419); Wing (30) to Ser (419); Phe (31) to Ser (419); Glu (32) to Ser (419); Ser (33) to Ser (419); Gly (34) to Ser (419); Leu (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); Wing (44) to Ser (419); Gly (45) to Ser (419); Glu (46) to Ser (419); Wing (47) to Ser (419); Thr (48) to Ser (419); Wing (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 f65) to Ser (419); Met (l), Glu (23), or Ala (24) to Met (418); Met (1), Glu (23), or Ala (24) to Gln (417); Met (1), Glu (23), or Ala (24) to Pro (416); Met (l), Glu (23), or Ala (24) to Arg (415); Met (l), Glu (23), or Ala (24) to Gln (414); Met (l), Glu (23), or Ala (24) to Trp (413); Met (l), Glu (23), or Ala (24) to Tyr (412); Met (l), Glu (23), or Ala (24) to Ser (411); Met (l), 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 (l), Glu (23), or Ala (24) to Arg (407); Met (l), Glu (23), or Ala (24) to Cys (406); Met (1), Glu (23), or Ala (24) to Val (405); Met (l), Glu (23), or Ala (24) to Glu (404); Met (l), Glu (23), or Ala (24) to Glu (403); Met (l). , Glu (23), or Ala (24) to Ser (402); Met (l), Glu (23), or Ala (24) to Gly (398); Met (l), Glu (23), or Ala (24) to Pro (397); Met (l), Glu (23), or Ala (24) to Lys (393); Met (l), Glu (23), or Ala (24) to Met (263); Met (l), Glu (23), or Ala (24) to Asp (311); Met (l), Glu (23), or Ala (24) to Pro (367); Met (l) to Ser (419); Met (l) to Ser (228); Glu (47) to Ser (419); Ala (III) to Lys (214); Wing (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 (llO) to Leu (215); Ala (lll) to Leu (215); Ala (112) to Leu (215); His (113) to Leu (215); Tyr (114) to Leu (215); Asn (115) to Leu (215); Thr (llß) to Leu (215); Thr (103) to Ser (228); Glu (104) to Ser (228); Glu (105) to Ser (228); Thr (106) to Ser (228); Ile (107) to Ser (228); Lys (108) to Ser (228); Phe (109) to Ser (228); Ala (llO) to Ser (228); Ma (lll) to Ser (228); Wing (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) to Arg (227); Glu (104) to Arg (227); Glu (105) to Arg (227); Thr (106) to Arg (227); Ile (107) to Arg (227); Lys (108) to Arg (227); Phe (109) to Arg (227); Ala (llO) to Arg (227); Ala (lll) to Arg (227); Wing (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 (llO) to Ser (213); Ala (lll) 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); Wing (110) to 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 (llO) to Leu (229); Ala (lll) to Leu (229); Ala (112) to Leu (229); His (113) to Leu (229); Tyr (114) to Leu (229); Asn (115) to Leu (229); Thr (llß) to Leu (229). Preferred embodiments include the following deletion mutations: Thr (103) -Arg (227); Glu (104) - Arg (227); Ala (112) - Arg (227); Thr (103) - Ser (213); Glu (104) - Ser (213); Thr (103) - Leu (215); Glu (47) - Ser (419); Met (l), Glu (23), or Ala (241-Met (263); Met (l), Glu (23), or Ala (24) - Asp (311); Met (l), Glu (23) ), or Ala (24) - Pro (367); Met (l) - Ser (419); and Met (l) - Ser (228) of (Figure 1 (SEQ ID NO: 18)) Also included by the present invention are the deletion mutations having amino acids deleted from both the NB terminus and the C terminus. Such mutations include all combinations of the N-terminal deletion mutations and the C-terminal deletion mutations described above. combinations can be made using recombinant techniques known to those skilled in the art, particularly, N-terminal deletions of the VEGF-2 polypeptide can be described by the general formula m-396, where m is an integer from -23 to 388 , where m corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. Preferably, the N-terminal deletions retain the area enclosed in the box, conserved from Figure 3 (PXCVXXXRCXGCCN) (SEQ ID NO: 8) and include polypeptides comprising the amino acid sequence of the residues: the N-terminal deletions of the polypeptide of the invention shown as SEQ ID NO: 1 include polypeptides that comprise the amino acid sequence of the residues: The a 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-ll to S-3.96; 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; S41 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-39 ~ 6; 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; 1-84 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; T- -95 to S- -396; L-96 to S-396; K-97 to S-396; S-98 to S-396; T-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; 1-165 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; 1-178 to S-396; S-179 to S-396; F-180 to S-396; A-181-a 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; 1-201 to S-396; 1-202 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 a ^ S ^ gß; 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 'to 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; 1-230 to S-396; C-231 to S-396; R-232 to S-396; C-233 to 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; 1-256 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-294 to S-396; V-295 to S-396; C-296 to S-396; K-297 to S-3B6; 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 to S-396; A-336 to S-396; C-337 to S-396; E-338 to S-396T 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-3"967 S-377 to S-396; Y-378 to S-396; S-379 to S-396; E-380 to S-396; E-381 to S-396; V -382 to S-396, C-383 to 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 of SEQ ID NO: 2. A preferred embodiment comprises amino acids S-205 to S.396 of the SEQ ID NO: 2. Polynucleotides encoding these polypeptides are also preferred.In addition, C-terminal deletions of the VEGF-2 polypeptide can also be described by the general formula -23-n, where n is an integer of - 15 to 395 where n corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. Preferably, the C-terminal deletions retain the area enclosed in the box, preserved from Figure 3 (PXCVXXXRCXGCCN) (SEC ID NO. 8), and include polypeptides comprising the amino acid sequence of the residues: Likewise, the C-terminal deletions of the polypeptide of the invention n shown as SEQ ID NO: 2 include polypeptides comprising the amino acid sequence of residues: E-l to M-395; E-l to Q-394; E-l to P-393; E-1 to R-392; E-l to Q-391; E-l to W-390; E-l to Y-389; E-1 to S-388; E-l to P-387; E-l to V-386; E-l to C-385; E-1 to R-384; E-1 to C-383; E-l to V-382; E-l to E-381; E-l to E-380; E-l to S-379; E-l to Y-378; E-1 to S-377; E-l to F-376; E-l to G-375; E-l to P-374; E-l to E-373; E-1 to C-372; E-l to A-371; E-l to K-370; E-l to Q-369; E-1 to R-368; E-l to N-367; E-l to T-366; E-l to C-365; E-l to P-364; E-1 to R-363; E-1 to R-362; E-l to Y-361; E-l to C-360; E-1 to S-359; E-1 to C-358; E-l to T-357; E-1 to Q-356; E-l to H-355; E-1 to H-354; E-1 to F-353; E-l to K-352; E-l to K-351; E-1 to 0-350; E-l to K- • 349; E-1 to L- -348; E-1 to L- -347; E-1 to C- -346; E-l to K- • 345; E-1 to Q-344; E-l to P-343; E-1 to S-342; E-l to E- -341; E-1 to T- -340; E-1 to C- -339; E-1 to E- -338; E-1 to C-337; E-l to A- 336; E-1 to C- -335; E-l to K- -334; E-l to G- • 333; E-1 to P- • 332; E-1 to N-331; He. 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-l to V- • 319; E-1 to C-318; E-l 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 F- -312; E-1 to F-3 1; 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-l to Q-304; E-1 to S-303; E-1 to P- -302; E-1 to F- - 301; E-1 to L- -300; E-l to K--299; E-l to N-298; E-l to K-297; E-1 to C- -296; E-1 to V- -295; E-1 to C- -294; E-l 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-l 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-l to A- 279; E-I to P-278; E-1 to R-277; E-1 to L--276; E-1 to G--275; E-l to A- -274; E-1 to R- -273; E-1 to C-272; E-l to V- • 271; E-1 to C--270; E-l to Q- -269; E-1 to C- -268; E-l to T- • 267; E-1 to E-266; E-l to E-265; E-1 to D-264; • E-l to L- -263; E-1 to E- -262; E-l to K- -261; E-l to N--260; E-l to P-259; E-1 to G- • 258; E-1 to C- -257; E-1 to T- -256; E-1 to D- -255; E-1 to H- -254; E-1 to F-253; E-l to G-252; E-1 to D-251; - E-l to T- -250; E-1 to S-249; E-1 to D-248; E-1 to D-247; E-l to G-246; E-l 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-l to F-239; E-1 to D-238; E-l to E- -237; E-l to Q- -236; E-1 to A-2 35; E-l 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-l to N- • 228; E-1 to N-227; E-1 to W-226; "E-1 to M-225; E-1 to Y-2 24; E-1 to N-223; E-1 to -222; E-1 to P-221; E-l to C-220; E-l to T- 219; E-l to K--218; E-1 to N--217; E-1 to A-216; E-l to A- • 215; E-1 to Q-214; E-1 to C-213; E-1 to Q-212; E-l to P- -211; E-1 to L--210; E-l to T- -209; E-l to A- -208; E-l 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-l 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-l to V-194; E-1 to D-193; E-1 to L--192; E-l to K- -191; E-1 to S-190; E-l to M- 89; E-1 to C-188; E-1 to R-187; E-1 to C-186; E-1 to S- -185; E-l to T- -184; E-1 to H- -183; E-1 to N- -182; E-l to A-181; E-l to F- • 180; E-1 to S- -179; E-1 to I- -178; E-1 to I- -177; E-1 to V-176; E-1 to P-175; E-l to K-174; E-1 to P-173; E-1 to G- -172; E-l to Q- -171; E-1 to S- -170; E-1 to L- -169; E-l 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-l to 1-161; E-l to K-160; E-1 to S- -159; E-1 to L--158; E-l 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-l to M- -151; E-1 to C-150; E-1 to Q-149; E-l to L-148; E-1 to G-147; • E-l to E- -146; E-1 to S- -145; E-1 to N- -144; E-1 to C- - 143; E-1 to C-142; E-l 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-l to P-132; E-1 to P-131; E-l to K-130; E-l to F-129; E-l to F-128; E-l to T-127; E-l to N-126; E-l to T-125; E-l to A-124; E-l to V-123; E-l to G-122; E-1 to F-121; E-l to E-120; E-l to K-119; E-l to G-118; E-l to V-117; E-1 to D-116; E-1 to 1-115; E-1 to C-114; E-l to V-113; E-l to E-112; E-l to R-III; E-l to P-110; E-l to M-109; E-1 to C-108; E-1 to Q-107; E-l to T-106; E-l to K-105; E-1 to R-104; E-l to W-103; E-l to E-102; E-l to N-101; E-l to D-100; E-1 to 1-99; E-1 to S-98; E-l to K-97; E-l to L-96; E-1 to 1-95; E-l to E-94; E-l to T-93; E-l to N-92; E-l to Y-91; E-l to H-90; E-l to A-89; E-l to A-88; E-1 to A-87: E-1 to F-86; E-l to K-85; E-1 to 1-84; E-1 to 1-83; E-l to E-82; E-l to E-81; E-l to T-80; E-l to R-79; E-1 to S-78; E-l to N-77; E-l to L-76; E-l to N-75; E-l to A-74; E-l to Q-73; E-l to E-72; E-1 to R-71; E-l to N-70; E-l to H-69; E-l to Q-68; E-l to W-67; E-1 to 0-66; E-1 to 0-65; E-l to K-64; E-l to R-63; E-l to L-62; E-l to Q-61; E-l to C-60; E-l to K-59; E-l to Y-58; E-l to M-57; E-l to K-56; E-l to W-55; E-l to Y-54; E-l to E-53; E-l to P-52; E-l to Y-51; E-l to L-50; E-l to V-49; E-l to T-148; E-l to M-47; E-l to L-46; E-l to E-45; E-l to D-44; E-l to V-43; E-1 to S-42; E-l to S-41; E-l to V-40; E-l to S-39; E-l to R-38; E-l to L-37; E-l to Q-36; E-l to E-35; E-l to E-34; E-l to L-33; E-l to D-32; E-l to K-31; E-l to 5-30; E-l to A-29; E-l to Y-28; E-l to A-27; E-1 to 1-26; E-l to A-25; E-l to E-24; E-l to G-23; E-l to A-22; E-l to D-21; E-l to P-20; E-l to E-19; E-l to A-18; E-1 to D-17; E-l to S-16; E-l to L-15; E-1 to D-14; E-l to L-13; E-l to G-12; E-l to S-ll; E-l to E-10; E-l to F-9; E-l to A-8; E-1 to A-7 of SEQ ID NO: 2. Polynucleotides encoding these polypeptides are also preferred. In addition, the invention also provides polypeptides having one or more amino acids deleted from both the amino and carboxyl termini, which can be described in general as having residues mn of SEQ ID NO: 2, where n and m are integers as described later. Likewise, deletions of the C-terminus of the VEGF-2 polypeptide of the invention shown as SEQ ID NO: 2 which includes polypeptides comprising the amino acid sequence of the residues: F-9 to M are also preferred. -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 8-388; 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 8-379, F-9 to Y-378, F-9 to 8-377, 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 N-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 Q-272; F-9 to V-271; F-9 to C-270; F-9 to Q-269; F-9 to C-268; F-9 to I- -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 1-256; 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 1-230, 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 I- -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-2 liF-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 1-178; F-9 to T-177; F-9 to V- -176; F-9 to P-175; F-9 to K- -174; 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-l 68; F-9 to V-l 67; F-9 to T-166; F-9 to 1-165; 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 1-115; F-9 to C-114; F-9 to V-113; F-9 to E-112; F-9 to R-III; 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 1-84; 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; of SEQ ID NO: 2. The polypeptide fragment comprising amino acid residues F-9 to R-203 of SEQ ID NO: 2, as well as also the polynucleotides that code for this polypeptide are specifically preferred. This F-9 to R-203 of the polypeptide of SEQ ID NO: 2 is preferentially associated with an S-205 to S-396 of the polypeptide of SEQ ID NO: 2. The association can be through disulfide, covalent or non-covalent interactions, by linking via a linker (e.g., serine, glycine, and proline linkages), or by an antibody. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through the sequence databases. Some of these sequences are related to SEQ ID NO-1 and may have been publicly available prior to the conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Listing each related sequence would be difficult. Accordingly, preferably excluded from the present invention are one or more nucleotides - comprising a nucleotide sequence described by the general formula of ab, while a is any integer from 1 to 1660 of SEQ ID NO: 1, b is an integer from 15 to 1674, where both a and b correspond to the positions of the nucleotide residues shown in SEQ ID NO: 1, and where b is greater than or equal to a + 14. Thus, in one aspect, provide N-terminal deletion mutations by the present invention. Such mutations include those comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 18) except for a deletion of at least the first 24 N-terminal amino acid residues (i.e. a deletion of at least Met (1) - Glu (24)) but not more than the first 115 N-terminal amino acid residues of Figure 1 (SEQ ID NO: 18). Alternatively, the first 24 N-terminal amino acid residues (ie, a deletion of at least Met (1) -Glu (24)) but not more than the first 103 N-terminal amino acid residues of Figure 1 ( SEQ ID NO: 18), etcetera. In another aspect, C-terminal deletion mutations are provided by the present invention. Such mutations include those comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 18) except for a deletion of at least the last C-terminal amino acid residue (Ser (419)) but not more than the latter 220 C-terminal amino acid residues (ie, a deletion of Val (199) -Ser amino acid residues (419)) of Figure 1 (SEQ ID NO: 18). Alternatively, the deletion will include at least the last C-terminal amino acid residue but not more than the last 216 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 18). Alternatively, the deletion will include at least the last C-terminal amino acid residue but no more than the last 204 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 18). Alternatively, the deletion will include at least the last C-terminal amino acid residues but not more than the last 192 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 18). Alternatively, the deletion will include at least the last C-terminal amino acid residues but not more than the last 156 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 18). Alternatively, the deletion will include at least the last C-terminal amino acid residues but not more than the last 108 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 18). Alternatively, the deletion will include at least the last C-terminal amino acid residues but not more than at least about 30 nt, and even more preferably, at least about 40 nt in length which are used as test substances of diagnosis and primers as discussed in the present. Naturally, the largest fragments 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, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650 or 1674 nt in length are also useful according to the present invention as are the fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA (s) or as shown in SEQ ID NO: SE SEQ ID NO: 3 For a fragment at least 20 nt in length, for example, fragments are proposed which include 20 or more contiguous bases of the nucleotide sequence of the deposited cDNA (s) or the nucleotide sequence as shown in SEQ ID NOS: lo 3. In addition, the representative examples of the fragments of polinuc Leotid of VEGF-2 include, for example, fragments having a sequence of approximately the number of nucleotides 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351 -400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950 or 951 at the end of SEQ ID NO: 1 or the cDNA contained in the deposited clone. In this context "approximately" includes the ranges particularly cited, larger or smaller by several nucleotides (5, 4, 3, 2, or 1), in either terminal or in both terminals. Preferably, these fragments encode a polypeptide which has biological activity. Fragments of the full-length gene of the present invention can be used as a hydridization test substance for a cDNA library to isolate the full-length cDNA and to isolate other cDNAs which have high sequence similarity to the gene or activity similar biological Test substances of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The test substance can also be used to identify a cDNA clone corresponding to a full length transcript and a clone or genomic clones containing the complete gene including regulatory and promoter regions, exons, and introns. An example of a selection comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide test substance. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to select a human cDNA library, genomic DNA or mRNA to determine which members of the library hybridize the test substance. A "polynucleotide" of VEGF-2 also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to the sequences contained in SEQ ID NO: 1 or for example, the contained cDNA clone (s) (s). ) in the deposits of the ATCC Nos. 97149 or 75698, the complement of the same. The "" stringent hybridization conditions "refer to an overnight incubation at 42 ° C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM phosphate potassium (pH 7.6), Denhardt 5x solution, 10% dextran sulfate, and 20 μg / ml denatured, cut or split salmon sperm DNA followed by washing the filters in 0.1 x SSC at approximately 65 ° C. Also contemplated are nucleic acid molecules that hybridize to VEGF-2 polynucleotides under stringent hybridization conditions.The changes in hybridization severity and signal detection are mainly realized through the manipulation of the formamide concentration. lower percentages of formamide result in the lowest severity); the salt conditions, or the temperature. For example, the lowest severity conditions include an overnight incubation at 37 ° C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl, 0.2M NaH2P04, 0.02M EDTA, pH 7.4), 0.5% SDS, formamide 30%, 100 ug / ml of salmon sperm blocking DNA; followed by washes at 50 ° C with SSPE IX; SDS 0.1%. In addition, to achieve even lower severity, washes made after rigorous hybridization can be made at higher salt concentrations (eg SSC 5X). Note that variations in the above conditions can be made through the inclusion and / or substitution of alternating blocking reagents used to suppress the environment in the hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available patented formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility. Of course, a polynucleotide which hybridizes only to the polyA + sequences (such as any polyA + 3 'terminal of a cDNA shown in the sequence listing), or to a complementary stretch of the T (or U) residues, would not be included in the definition of "polynucleotide", since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (eg, virtually any clone of double-stranded cDNA or strand). For a polynucleotide which hybridizes to a "portion" of a polynucleotide is proposed a polynucleotide (either DNA or RNA) that hybridizes to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, even more preferably at least about 30 nt , and even much more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic test substances and primers as discussed above and in more detail later.

For a portion of a polynucleotide of "at least 20 nt in length", for example, 20 or more contiguous nucleotides of the nucleotide sequence of the reference polynucleotide are proposed (eg, the deposited cDNA or the nucleotide sequence as described). shows in SEQ ID NO: l). Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the poly (A) 3 N terminal of the VEGF-2 cDNA shown in SEQ ID NOS: 1 or 3), or to a complementary stretch of T residues ( or U), would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since this nucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement of the same (for example, practically any double-stranded or strand cDNA clone). The present application is directed to nucleic acid molecules at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NOS: 1 or 3 or to the nucleic acid sequence of the (c) deposited cDNA (s), regardless of whether they encode a polypeptide having VEGF-2 activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having VEGF-2 activity, one of skill in the art would still know or use the nucleic acid molecule, for example, as a test substance. of hybridization or a polymerase chain reaction (PCR) primer. The uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having VEGF-2 activity include, inter alia, (1) isolating the VEGF-2 gene or allelic variants thereof in a gene library of CDNA; (2) hybridization in itself (eg, "FISH") for the metaphase chromosomal extensions to provide chromosomal location requires the VEGF-2 gel, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and analysis with Northern blotting paper to detect the expression of VEGF-2 mRNA in specific tissues. However, nucleic acid molecules having at least 95%, 96%, 97%, 98% or 99% sequences identical to a nucleic acid sequence shown in SEQ ID NOS: 1 or 3 or to a sequence of nucleic acids are preferred. nucleic acid of the deposited cDNA (s), which (s), in fact, encodes a polypeptide having VEGF-2 protein activity. By "a polypeptide having VEGF-2 activity" polypeptides are proposed which exhibit VEGF-2 activity in a particular biological assay. For example, the protein activity of VEGF-2 can be measured using, for example, mitogenic assays and endothelial cell migration assays. - See, for example, Olofsson et al., Proc. Na ti. Acad. Sci. USA 93: 251 6-2581 (1996) and Joukov et al., EMBO J. 5: 290-298 (1996). Naturally, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence of at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequence of the deposited cDNA (s) or the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 will encode a polypeptide "having VEGF-2 protein ". In fact, since the degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the comparison test described above. It will further be recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having VEGF-2 protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to effect the protein function significantly (eg, the replacement of an aliphatic amino acid with a second aliphatic amino acid). For example, guidance on how to make phenotypically inactive amino acid substitutions is provided in Bowie, JU et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Sci en 24 7: 1306-1310 (1990). , where the authors indicate that proteins are surprisingly tolerant of amino acid substitutions. In this manner, the present invention is directed to polynucleotides having an identity of at least 70%, preferably at least 90% and most preferably an identity of at least 95%, 96%, 97% or 98% a a polynucleotide which encodes the polypeptides of SEQ ID NOS: 2 or 4, as well as fragments thereof, fragments having at least 30 bases and preferably at least 50 bases and the polypeptides encoded by these polynucleotides. "Identity" per se has a recognized significance in the art and can be calculated using the last 52 C-terminal amino acid residues of Figure 1 (SEQ ID NO: 18). In yet another aspect, also included by the present invention are the deletion mutations having amino acids deleted from both the N-terminal and C-terminal residues. Such mutations include all combinations of the N-terminal deletion mutations and the C-terminal deletion mutations described above. The term "gene" means the segment of DNA involved in the production of a polypeptide chain; this includes the preceding and following regions of the coding region (leader and trailer) as well as the intervening sequences (introns) between the individual coding segments (exons). The present invention is further directed to fxagments of the isolated nucleic acid molecules described herein. For a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA (s) or the nucleotide sequence shown in SEQ ID NO: SEQ ID NO: 3 are proposed fragments of at least about 15 nt, and more preferably at least about 20 nt, even more preferably published techniques. (See, for example: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, AM, ed., Oxford University Press, New York, (1988), BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, DW, ed., Academic Press, New York, ( 1993); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, AM, and Griffin, HG, 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).) While there are a number of methods for measuring the identity between two polynucleotide sequences or polypeptide, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Appl i ed Ma th. 48: 101 3 (1988)). determining the identity or similarity between two sequences include, but are not limited to, those described in "Guide to Huge Computers," Martin J. Bishop, ed., Academic Press, San Diego, (1994), and Carillo, H., and Lipton, D., SIAM J. Appl i ed Ma th. 48: 101 3 (1988). Methods for aligning polynucleotides or polypeptides are encoded in computer programs, including the GCG program package (Devereux, J., et al., Nucl ei c Acids Review 12 (1): 381 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Molec. Biol. 225: 403 (1990), Bestfit program (Wisconsin Sequence Analysis Package, Version 8 by Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WO 53711 (using the local homology algorithm of Smith and Waterman, Advan ces in Applied Ma thema ti cs 2: 4 82 -489 (1981)). For a polynucleotide having at least one nucleotide sequence, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is proposed that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five dot mutations per 100 nuc leotides of the reference nucleotide sequence encoding the VEGF-2 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleophilic sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the complete nucleotides in the reference sequence can be inserted into the reference sequence. The sequence in question may be a complete sequence SEQ ID NO: 1, the ORF (open reading frame), or any specified fragment as described herein. As a practical matter, if any particular nucleic acid molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention it can be determined conventionally using known computer programs. A preferred method for determining the best total binding between a sequence in question (a sequence of the present invention) and an objective sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 5: 237-245 (1990)). In a sequence alignment, the sequences in question and objective are both DNA sequences. An RNA sequence can be compared by converting U 's to T's. The result of global sequence alignment is in the percentage of identity. The preferred parameters used in a FASTDB alignment of the DNA sequences to calculate the percent identity are: Matrix = Unitary, k-tuple = 4, Inequality Penalty = l, Penalty for Union = 30, Length of Randomization Group = 0 , Court Record = l, Penalty for Opening = 5, Penalty for Size of the Opening 0.05, Window Size = 500 or the length of the target nucleotide sequence, whichever is shorter. If the target sequence is shorter than the sequence in question due to 5 'or 3' deletions, not due to internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not take into account the truncations 5 ', 3' of the target sequence when calculating the percentage of identity. For the target sequences truncated at the 5 'or 3' ends, relative to the sequence in question, the percent identity is corrected by calculating the number of bases of the sequence in question that are 5 'and 3' of the target sequence, which is not linked / aligned, as a percentage of the total bases of the sequence in question. If a nucleotide is bound / aligned it is determined by the results of the alignment of the FASTDB sequence. This percentage is then subtracted from the percentage of identity, calculated by the previous FASTDB program using the specified parameters, to arrive at a record of the percentage of final identity. This corrected record is what is used for the purposes of the present invention. Only the bases outside the 5 'and 3' bases of the target sequence, as visually represented by the alignment of the FASTDB, which are not linked / aligned with the sequence in question, are calculated for the purposes of manual adjustment of the record of the percentage of identity. For example, a target sequence of 90 bases is aligned to a sequence in question of 100 bases to determine the percent identity. Deletions occur at the 5 'end of the target sequence and therefore, alignment of the FASTDB does not show a binding / alignment of the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5 'and 3' unbound / total number of bases in the sequence in question) so that 10% is subtracted from the percent identity record calculated by the FASTDB program. If the remaining 90 bases fit together perfectly, the final identity percentage would be 90%. In another example, a target sequence of 90 bases is compared to a sequence of 100 bases. This time the deletions are internal deletions so that they are not bases at the base 5 'or 3' of the target sequence which are not linked / aligned with the sequence in question. In this case, the identity percentage calculated by the FASTOB is not corrected manually. Again, only the 5 'and 3' bases of the target sequence which are not bound / aligned with the sequence in question are manually corrected. No other manual corrections are made for the purposes of the present invention. For a polypeptide having at least one amino acid sequence, for example 95% "identical" to a subject amino acid sequence of the present invention, it is proposed that the amino acid sequence of the target polypeptide be identical to the sequence in question, except that the target polypeptide sequence can include up to five amino acid alterations per 100 amino acids of the amino acid sequence in question. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a sequence of amino acids in question, 'up to 5% of the amino acid residues in the target sequence can be inserted, suppress, (indels) or replace with another amino acid. These alterations of the reference sequence may occur at the terminal amino or carboxy positions of the reference amino acid sequence or any between those terminal positions, interspersed either individually between the residues in the reference sequence or in one or more contiguous groups within the reference sequence. As a practical matter, if any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the amino acid sequences shown in the Table loa the amino acid sequence encoded by the deposited DNA clone can be determined conventionally using known computer programs- A preferred method for determining the best total binding between a sequence in question (a sequence of the present invention) and an objective sequence, also referred to as a global sequence alignment , can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp.App.Biosci. (1990) 5: 237-245). In a sequence alignment the sequences in question and target are both either nucleotide sequences or both amino acid sequences. The result of the global alignment of sequences is in percentage of identity. The preferred parameters used in an amino acid alignment of the FASTDB are: Matrix-PAM 0, k-tuple = 2, Penalty for Inequality = l, Penalty for Union = 20, Length of the Randomization Group = 0, Court Record = l, Window size = sequence length, Opening Penalty = 5, Penalty for Opening Size = 0.05, Window Size = 500 or the length of the target amino acid sequence, whichever is shorter. If the target sequence is shorter than the sequence in question due to N- or C-terminal deletions, not due to internal deletions, a manual correction to the results must be made. This is because the FASTDB program does not take into account the N- and C-terminal truncations of the target sequence when calculating the overall identity percentage. For the target sequences truncated at terminals N and C, relative to the sequence in question, the percent identity is corrected by calculating the number of residues of the sequence in question that are N- and C-terminal of the target sequence, the which are not bound / aligned with a corresponding target residue, as a percentage of the total bases of the sequence in question. If a residue is joined / aligned it is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percentage of identity, calculated by the previous FASTDB program using the specified parameters, to arrive at a record of the percentage of final identity. This record of the final identity percentage is what is used for the purposes of the present invention. Only the residuals for the N and C endings of the target sequence, while not bound / aligned with the sequence in question, are considered for manual adjustment purposes of the percent identity record. That is, only the positions of the residues in question outside the N- and C-terminal residues more remote from the target sequence. For example, an objective sequence of 90 amino acid residues is aligned with a sequence of 100 residues to determine the percent identity. Deletion occurs at the N-terminus of the target sequence and therefore, the alignment of the FASTDB does not show a binding / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (the number of residues in the unbound N and C endings / the total number of residues in the sequence in question) so that 10% of the identity percentage record calculated by the FASTDB program is subtracted. If the remaining 90 residues were perfectly joined, the final identity percentage would be 90%. In another example, an objective sequence of 90 residues is compared to a sequence in question of 100 residues.

This time the deletions are internal deletions so that there are no residues in the N or C endings of the target sequence which are not linked / aligned with the sequence in question. In this case, the identity percentage calculated by the FASTDB is not corrected manually. Again, only the positions of the residues outside the N- and C-terminal ends of the target sequence, as visually represented in the alignment of the FASTDB, which are not bound / aligned with the sequence in question are manually corrected . No other manual corrections are made for the purposes of the present invention.

VEGF-2 Polypeptides The present invention further relates to polypeptides which have the amino acid sequence deduced from Figures 1 or 2, or which have the amino acid sequence encoded by the deposited cDNAs, as well as fragments, analogs and derivatives of such polypeptides. The terms "fragment", "derivative" and "analogue" when referring to the polypeptide of Figures 1 or 2 or that encoded by the deposited cDNA, mean a polypeptide which retains the conserved configuration of the VEGF proteins as shown in FIG. Figure 3 and essentially the same biological function or activity. In the present invention, a "polypeptide fragment" refers to a short amino acid sequence contained in SEQ ID NO: 2 or encoded by the cDNA contained in the deposited clone. The protein fragments may be "free-standing", or comprised within a larger polypeptide of which the fragment forms a part or region, more preferably as a single, continuous region. Representative examples of polypeptide fragments of the invention include, for example, fragments of about a number of amino acids 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-¿80, 181-200, 201-220, 221-240, 241-260, 261-280 or 281 at the end of the coding region. In addition, polypeptide fragments may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 amino acids in length. In this context "approximately" includes the ranges particularly cited, larger or smaller by several amino acids (5, 4, 3, 2 or 1), at either end or at both ends. Preferred polypeptide fragments include the secreted VEGF-2 protein as well as the fully developed form. In addition, preferred polypeptide fragments include the secreted VEGF-2 protein or the fully developed form having a continuous series of deleted residues of the amino terminus or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted VEGF-2 polypeptide or the fully developed form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted VEGF-2 protein or fully developed form. In addition, any combination of the deletions of the above amino or carboxy termini is preferred. Similarly, fragments of polynucleotides encoding these VEGF-2 polypeptide fragments are also preferred. Also preferred are fragments of VEGF-2 polypeptides and polynucleotides characterized by structural or functional domains, such as the fragments comprising the alpha-helix and the regions forming the alpha-helix, the beta-sheet and the regions forming the alpha-helix. beta-sheet, turn and the regions forming a turn, a spiral and the regions forming a spiral, hydrophilic regions, hydrophobic regions, alpha antipathetic regions, unfriendly beta regions, flexible regions, forming regions the surface, the binding region of the substrate and the regions of high antigenic index. Fragments of polypeptides of SEQ ID NO: 2 that are within conserved domains are specifically contemplated by the present invention. (See Figure 2). further, fragments of polynucleotides encoding these domains are also contemplated. Other preferred fragments are the biologically active VEGF-2 fragments. The biologically active fragments are those that exhibit activity similar, but not necessarily identical, to an activity of the VEGF-2 polypeptide. The biological activity of the fragments may include a desired, improved activity, or an undesirable, decreased activity. The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides. It will be recognized in the art that some amino acid sequences of the VEGF-2 polypeptide can be varied without a significant effect of the structure or function of the protein. If these differences in the sequence are contemplated, it must be remembered that there will be critical areas in the protein which determine the activity. Thus, the invention further includes variations of the VEGF-2 polypeptide which exhibit substantial VEGF-2 polypeptide activity or which include regions of the VEGF-2 protein such as the portions of the protein discussed above. These mutations include deletions, insertions, inversions, repetitions and substitutions of types. As indicated above, guidance regarding which amino acid changes are likely to be phenotypically inactive can be found in Bo ie, J.U. and collaborators, "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306-1310 (1990). In this manner, fragments, derivatives or analogs of the polypeptides of Figures 1 or 2, or that encoded by the deposited cDNAs can be: (I) one in which one or more of the amino acid residues are substituted with a residue of conserved or non-conserved amino acids (preferentially a conserved amino acid residue) and this substituted amino acid residue may or may not be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the fully developed polypeptide is fused with another compound, such as a compound to increase the life span of the polypeptide (eg, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the fully developed polypeptide, such as a leader or secretory sequence or a sequence which is used for the purification of the fully developed polypeptide or a sequence of proproteins; or (v) one in which it comprises a few amino acid residues shown in SEQ ID NO: 2 or 4, and retains the conserved configuration and still retains the characteristics of the activity of the VEGF family of the polypeptides. Such fragments, derivatives and analogs are considered to be within the scope of those skilled in the art of the teachings herein. Substitutions of charged amino acids with charged Qtro amino acid and with neutral or negatively charged amino acids are of particular interest. The latter result in proteins with positive charge, reduced to improve the characteristics of the VEGF-2 protein. The prevention of aggregation is highly desirable. The aggregation of the proteins not only results in a loss of activity but can also be a problem when preparing pharmaceutical formulations, because these can 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. Therapeu ti c Drug Carrier Sys tems 1 0: 301-311 (1993)). Amino acid replacement can also change the selectivity of binding to receptors on the surface of cells. Ostade et al., Na ture 361: 266-268 (1993) describes certain mutations that result in the selective binding of TNF-a to only one of the two known types of TNF receptors. Thus, the VEGF-2 of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or by human manipulation. As indicated, the changes are preferably of a minor nature, such as conservative amino acid substitutions ... which do not significantly affect the folding or activity of the protein (see Table 1).

TABLE 1. Conservative Amino Acid Substitutions Naturally, the number of amino acid substitutions that a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of substitutions for any given VEGF-2 polypeptide will not be greater than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

The amino acids in the VEGF-2 protein of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-1085 (1989)). The last procedure introduces individual alanine mutations in each residue in the molecule. The resulting mutant molecules are then subjected to a biological activity test such as receptor binding or proliferative activity in vi tro, or in vivo. Sites that are critical for binding to the ligand receptor can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al J. Mol. Biol. 224: 8 99-904 (1992 ) and de Vos et al. Sci en 255: 306-312 (1992)). The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified for homogeneity. The term "isolated" means that the material is separated from its original environment (for example, the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such a polynucleotide could be part of a vector and / or this polynucleotide or polypeptide could be part of a composition, and even be isolated in that such vector or composition is not part of its natural environment. - In specific embodiments, the 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 a further embodiment, the polynucleotides of the invention comprise at least 15 contiguous nucleotides of the coding sequence of VEGF-2, but do not comprise all or a portion of some intron of VEGF-2. In another embodiment, the nucleic acid comprising the coding sequence of VEGF-2 does not contain the coding sequences of a flanking, genomic (i.e., 5 'or 3' gene for the VEGF-2 gene in the genome). The polypeptides of the present invention include the polypeptides of SEQ ID NOS: 2 and 4 (in particular the fully developed polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptides of SEQ ID NOS: 2 and 4, and more preferably at least 90% similarity (most preferably at least 95% identity) to the polypeptides of SEQ ID NOS: 2 and 4 and to a more preferably at least 95% similarity (even more preferably at least 90% identity) to the polypeptides of SEQ ID NOS: 2 and 4 and also include portions of such polypeptides with such a portion of the polypeptide containing generally at least 30 amino acids and more preferably at least 50 amino acids. As is known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substituents of a polypeptide to the sequence of a second polypeptide. The fragments or portions of the polypeptides of the present invention can be used to produce the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments can be used as intermediates to produce the full-length polypeptides. The fragments or portions of the polynucleotides of the present invention can be used to synthesize the full length polynucleotides of the present invention. The polypeptides of the present invention include the polypeptide encoded by the deposited cDNA including the leader; the fully developed polypeptide, encoded by the deposited cDNA minus the leader (ie, the fully developed protein); a polypeptide comprising amino acids from about -23 to about 396 in SEQ ID NO: 2; a polypeptide comprising amino acids from about -22 to about 396 in SEQ ID NO: 2; a polypeptide comprising amino acids from about 1 to about 396 in SEQ ID NO: 2; as well as also polypeptides which are at least 95% identical, and most preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

Fusion Proteins Any VEGF-2 polypeptide can be used to generate the fusion proteins. For example, the VEGF-2 polypeptide, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the VEGF-2 polypeptide can be used to "indirectly" detect the second protein by binding to VEGF-2, in addition, because the secreted proteins target cell sites based on traffic signals, VEGF-2 polypeptides can be used as target or target molecules once fused with other proteins Examples of domains that can be fused to VEGF-2 polypeptides include not only heterologous signal sequences, but also other functional regions, The fusion does not essentially need to be direct, but can occur through linker sequences.Furthermore, the fusion proteins can also be designed to improve the characteristics of the VEGF-2 polypeptide, for example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the polypeptide of VEGF-2 to improve stability and persistence during purification of the host cell or subsequent handling and storage. Also, portions of peptides can be added to the VEGF-2 polypeptide to facilitate purification. Such regions can be separated before the final preparation of the VEGF-2 polypeptide. The addition of the peptide portions to facilitate the handling of the polypeptides are familiar and routine techniques in the art. In addition, the VEGF-2 polypeptides, including the fragments, and specifically the epitopes, can be combined with portions of the constant domain of immunoglobulins (IgG), which results in chimeric polypeptides. These fusion proteins facilitate purification and show an increased life period in vivo. One reported example describes the chimeric proteins consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (European Patent Application No. 394,827; Traunecker et al., Na ture 331: 84-8 (1988)). Fusion proteins that have dimeric structures linked to disulfide (due to IgG) may also be more efficient at binding and neutralizing other molecules, than the secreted, monomeric protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270: 3958-3964 (1995)). Similarly, European Patent Application No. 0 464 533 (Canadian counterpart 2045869) describes the fusion proteins comprising various proteins of the constant region of the immunoglobulin molecules together with another human protein or a part thereof. In many cases, the Fe part in a fusion protein is beneficial in therapy and diagnosis, and thus may result, for example, in improved pharmacokinetic properties, / European patent application No. 0232 262). Alternatively, it would be desired to suppress the Fe part after the fusion protein has been expressed, deleted and purified. For example, the Fe moiety may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fe portions for the purpose of high throughput screening assays to identify hIL-5 antagonists. (See, D. Bennett et al., J. Molecular Recognition 8: 52-58 (1995); K. Johanson et al., J. Bi ol. Chem. 270: 9459-9411 (1995)). In addition, the VEGF-2 polypeptides can be fused to the marker sequences, such as a peptide which facilitates the purification of VEGF-2. In the preferred modalities, the marker amino acid sequence is a hexa-histidine peptide, such as a label provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chats orth, CA 91311), among others, many of which are commercially available . As described in Gentz et al., Proc. Na ti. Acad. Sci, USA 86: 821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37: 1 61 (1984)). In this way, any of those above fusions can be designed using the polynucleotides or VEGF-2 polypeptides.

Biological Activities of VEGF-2 Polynucleotides and VEGF-2 polypeptides can be used in assays to analyze one or more biological activities. If VEGF-2 polynucleotides and polypeptides exhibit activity in a particular assay, it is likely that VEGF-2 may be involved in diseases associated with biological activity. Therefore, VEGF-2 could be used to treat the associated disease.

Immune Activity Polypeptides or polynucleotides of VEGF-2 may be useful in the treatment of deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid cells (platelets, red blood cells, neutrophils and macrophages) and lymphoid cells (B and T lymphocytes) of pluripotent stem cells. The etiology of these immune deficiencies or disorders can be genetic, somatic, such as cancer or some autoimmune disorders, acquired (for example, by chemotherapy or toxins), or infectious. In addition, VEGF-2 polynucleotides or polypeptides can be used as markers or detectors of a particular disease or disorder of the immune system. The VEGF-2 polynucleotides or polypeptides may be useful in the treatment or detection of hematopoietic cell deficiencies or disorders. Polynucleotides or VEGF-2 polypeptides could be used to increase the differentiation and proliferation of hematopoietic cells, including pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types of hematopoietic cells. . Examples of immune deficiency syndromes include, but are not limited to: blood protein disorders (eg, agammaglobulinemia, disgammaglobulinemia), telangiectasia ataxia, common variable immunodeficiency, Digeorge syndrome, HIV infection, HTLV-BLV infection, syndrome of adhesion deficiency of leukocytes, lymphopenia, phagocytic bactericidal dysfunction, combined immunodeficiency, severe (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia or hemoglobinuria. In addition, VEGF-2 polypeptides or polynucleotides can also be used to modulate hemostatic activity (the arrest of bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostatic or thrombolytic activity, polynucleotides or VEGF-2 polypeptides could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders ( for example thrombocytopenia), or injuries resulting from trauma, surgery or other causes. Alternatively, polynucleotides or VEGF-2 polypeptides that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve coagulation, important in the treatment of heart attacks (infarction), strokes, or scarring. The polynucleotides or polypeptides of the VEGF-2 may also be useful in the treatment or detection of autoimmune disorders. Many autoimmune disorders result from the inappropriate recognition of themselves as a foreign material by immune cells. This inappropriate recognition results in an immune response that leads to the destruction of the host tissue. Therefore, the administration of VEGF-2 polypeptides or polynucleotides that can inhibit an immune response, particularly the proliferation, differentiation or chemotaxis of T cells, can be an effective therapy in the prevention of autoimmune disorders. Examples of autoimmune disorders that can be treated or detected by VEGF-2 include, but are not limited to: Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture Syndrome, Graves Disease, Multiple Sclerosis, Grave Myasthenia, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purple, Reiter's Disease, Stiff Syndrome Man, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitus and inflammatory eye disease, autoimmune. Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, can also be treated by VEGF-2 polypeptides or polynucleotides. In addition, VEGF-2 can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility. The polynucleotides or the polypeptides of VEGF-2 can also be used to treat and / or prevent organ rejection or graft-versus-host disease (GVHD). The rejection of organs occurs by the destruction of immune cells, host of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, foreign, transplanted immune cells destroy host tissues. The administration of VEGF-2 polypeptides or polynucleotides that inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T cells, can be an effective therapy in the prevention of organ rejection or GVHD. Similarly, VEGF-2 polypeptides or polynucleotides can also be used to modulate inflammation. For example, VEGF-2 polypeptides or polynucleotides can inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (eg, septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury , endotoxin lethality, arthritis, hyperacute complement-mediated rejection, nephritis, lung injury induced by cytokine or chemokine, inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines (eg, TNF or IL-). 1) .

Hyperproliferative disorders Polypeptides or polynucleotides of the VEGF-2 can be used to treat or detect hyperproliferative disorders, including neoplasms.

The VEGF-2 antagonist polypeptides or polynucleotides can inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, VEGF-2 antagonist polypeptides or polynucleotides can proliferate other cells which can inhibit hyperproliferative disorder. For example, by increasing an immune response, particularly increasing the antigenic qualities of the hyperproliferative disorder or by proliferation, differentiation or mobilization of T cells, hyperproliferative disorders can be treated. This immune response can be increased either by increasing an existing immune response, or by initiating a new immune response. Alternatively, the decrease in an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent. Examples of hyperproliferative disorders that can be treated or detected by polynucleotides or VEGF-2 antagonist polypeptides include, but are not limited to, neoplasms located in: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testes, ovary, thymus, thyroid), eye, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, vessel, thoracic and urogenital. Similarly, other hyperproliferative disorders can also 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, paraproteinemias, purpura, sarcoidosis, Sezary's syndrome, Waldenstron's macroglobulinemia, Gaucher's disease, histiocytosis, and any other hyperproliferative disease, in addition to neoplasia, localized in an organ system listed above.

Infectious Disease Polypeptides or VEGF-2 polynucleotides can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and / or T cells, infectious diseases can be treated. The immune response can be increased either by increasing an existing immune response, or by initiating a new immune response. Alternatively, VEGF-2 polypeptides or polynucleotides can also directly inhibit infectious agents, without necessarily producing an immune response. Viruses are an example of an infectious agent that can cause a disease or symptoms that can be treated or detected by VEGF-2 polynucleotides or polypeptides. Examples of viruses include, but are not limited to, the following viral families of DNA and RNA: Arboviruses, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (for example, Paramixoviridae, Morbillivirus, Rabdoviridae), Ortomixoviridae (for example, Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vacinia), Reoviridae (for example, Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus) and Togaviridae (for example, Rubivirus). Viruses found within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiolitis, encephalitis, eye infections (eg, conjunctivitis, keratitis), fatigue syndrome chronic, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (eg, AIDS), pneumonia, Burkitt's lymphoma, chicken pox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold common, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (eg, sarcoma, warts), and viremia. The VEGF-2 polypeptides or polynucleotides can be used to treat or detect any of these symptoms or diseases. Similarly, bacterial or fungal agents that can cause a disease or symptoms and that can be treated or detected by poly-nucleotides or VEGF-2 polypeptides include, but are not limited to, the following Gram-Negative and Gram bacterial families. -Positive and fungal: Actinomycetales (eg, Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacilaceae (eg, Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacteria, Coccidioidomycosis, Cryptococcosis, Dermatocicosis, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacteria, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceas (for example, Acinetobacteria, Gonorrhea, Menigococcal), Pasteurellaceae Infections (for example, Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamidiaceas, Syphilis, and Staphylococcal. These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (eg, infections related to the AIDS), paronychia, prosthetic-related infections, Reiter's disease, respiratory tract infections, such as whooping cough or empyema, sepsis, Lyme disease, cat scratch disease, dysentery, parasite fever, food poisoning, typhoid, pneumonia, gonorrhea, Meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic fever, Scarlet fever, sexually transmitted diseases, skin diseases (eg, cellulitis, dermatocicosis), toxemia, infections of the urinary tract, wound infections. The VEGF-2 polypeptides or polynucleotides can be used to treat or suppress any of these symptoms or diseases. In addition, parasitic agents that cause a disease or symptoms that can be treated or detected by VEGF-2 polynucleotides or polypeptides include, but are not limited to, the following families: Amibiasis, Babesiosis; Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Durina, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis and Trichoas. These parasites can cause a variety of diseases or symptoms, including, but not limited to: scabies, thrombiculiasis, eye infections, intestinal disease (eg, dysentery, giardiasis), liver disease, lung disease, opportunistic infections (for example, related to AIDS), Malaria, complications of pregnancy and toxoplasmosis. The VEGF-2 polypeptides or polynucleotides can be used to treat or detect any of these symptoms or diseases. Preferably, the treatment using the VEGF-2 polypeptides or polynucleotides could be either by administering an effective amount of the VEGF-2 polypeptide to the patient, or by removing the patient's cells, supplying the cells with the VEGF-2 polynucleotide, and the return of the designed cells to the patient (ex vivo therapy). In addition, the VEGF-2 polypeptide or polynucleotide can be used as an antigen in a vaccine to raise an immune response against an infectious disease.

Regeneration Polynucleotides or VEGF-2 polypeptides can be used to differentiate, proliferate and attract cells, leading to the regeneration of tissues. (See, Sci en 276: 59-81 (1997)). Tissue regeneration could be used to repair, replace or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (eg osteoporosis, osteoarthritis, periodontal disease, liver failure) surgery, including plastic surgery, cosmetics, fibrosis, reperfusion injury, or damage to systemic cytokines. The tissues that could be regenerated using the present invention include organs (eg, pancreas, liver, intestine, kidney, skin, endothelium), muscle tissue (smooth, skeletal or cardiac), vascular (including vascular endothelium), lymphatic (including the lymphatic endothelium), nervous, hematopoietic and skeletal (bone, cartilage, tendon, and ligament). Preferably, regeneration occurs without or with diminished healing. Regeneration may also include angiogenesis. In addition, polynucleotides or VEGF-2 polypeptides can increase the regeneration of difficult-to-cure tissues. For example, increased regeneration of the tendon / ligament would accelerate the recovery time after damage. The VEGF-2 polynucleotides or polypeptides of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. An additional example of tissue regeneration from wounds that do not heal includes pressure ulcers, ulcers associated with vascular insufficiency, surgery and traumatic injuries. Similarly, nerve and brain tissue could also be regenerated by using VEGF-2 polynucleotides or polypeptides to proliferate and differentiate nerve cells. Diseases that could be treated using this method include diseases of the central and peripheral nervous system, neuropathies, or mechanical and traumatic diseases (eg, spinal cord disorders, head trauma, cerebrovascular disease, and stroke). Specifically, diseases associated with legions of peripheral nerves, peripheral neuropathy (eg, resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (eg, Alzheimer's disease, Parkinson's disease, of Huntington, amyotrophic lateral sclerosis, and Shy-Drager syndrome), all could be treated using polynucleotides or VEGF-2 polypeptides.

Chemotaxis Polynucleotides or polypeptides of VEGF-2 can have chemotactic activity. A chemotactic molecule attracts or mobilizes cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells or Mastzelle, eosinophils, epithelial and / or endothelial cells) to a particular site in the body, such as inflammation, infection or a hyperproliferation site. The mobilized cells can then reject and / or cure the particular trauma or abnormality.

Polynucleotides or VEGF-2 polypeptides can increase the chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders or any disorder of the immune system by increasing the number of cells that target or target a particular location in the body. For example, chemotactic molecules can be used to treat wounds and another to tissues by attracting immune cells to the injured location. When a chemotactic molecule, VEGF-2 could also attract fibroblasts, which can be used to treat wounds. It is also contemplated that polynucleotides or VEGF-2 polypeptides can inhibit chemotactic activity. These molecules could also be used to treat disorders. In this manner, VEGF-2 polynucleotides or polypeptides could be used as an inhibitor of chemotaxis.

Binding Activity VEGF-2 polypeptides can be used for the selection of molecules that bind to VEGF-2 or molecules to which VEGF-2 binds. The binding of VEGF-2 and the molecule can activate (agonist), increase, inhibit (antagonist), or decrease the activity of VEGF-2 or the bound molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors) or small molecules. Preferably, the molecule is closely related to the natural ligand of VEGF-2, for example, a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Curren t Protocols in Immunolgy 1 (2): Chapter 5 (1991)). Similarly, the molecule may be closely related to the natural receptor to which VEGF-2 binds (ie, Flt-4), or at least, a fragment of the receptor capable of being bound by VEGF-2 (e.g. , an active site). In any case, the molecule can be rationally designed using known techniques. Preferably, the selection for these molecules involves the production of the appropriate cells which express VEGF-2, either as a secreted protein or in the cell membrane. Preferred cells include mammalian, yeast, Drosophila or E. coli cells. Cells expressing VEGF-2 (or the cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound that potentially contains the molecule to observe binding, stimulation, or inhibition of the activity of either the VEGF-2 or the molecule. The assay can simply analyze the binding of a candidate compound to VEGF-2, where the binding is detected by a tag, or in an assay that involves competition with a tagged competitor. In addition, the assay can analyze whether the candidate compound results in a signal generated by binding to VEGF-2. Alternatively, the assay can be carried out using the cell-free preparations, a polypeptide / molecule attached to a solid support, chemical libraries, or mixtures of natural products. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing VEGF-2, measuring the activity or binding of VEGF-2 / molecule and comparing the activity or binding of VEGF-2 / molecule to a model. Preferably, an ELISA assay can measure the level or activity of VEGF-2 in a sample (eg, a biological sample) using a monoclonal or polyclonal antibody. The antibody can measure the level or activity of VEGF-2 either by binding, directly or indirectly, to VEGF-2 or by competing with VEGF-2 for a substrate. All these previous assays can be used as diagnostic or prognostic markers. The discovered molecules using these assays can be used to treat a disease or to cause a particular result in a patient (e.g., development or growth of blood vessels) by activation or inhibition of the VEGF-2 / molecule. In addition, the assays can discover agents that can inhibit or increase the production of VEGF-2 from properly manipulated cells or tissues. - Therefore, the invention includes a method for identifying compounds which bind to VEGF-2 comprising the steps of: (a) incubating a candidate binding compound with VEGF-2; and (b) determine if the union has occurred. In addition, the invention includes a method for identifying agonists / antagonists comprising the steps of: (a) incubating a candidate compound with VEGF-2, (b) subjecting a test to biological activity, and (b) determining whether it has A biological activity of VEGF-2 has been altered.

Other Activities Polypeptides or polynucleotides of VEGF-2 can also increase or decrease the differentiation or proliferation of embryonic stem cells, in addition to, as discussed previously, the hematopoietic lineage. VEGF-2 polypeptides or polynucleotides can also be used to modulate the characteristics of mammals, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size and shape (eg. example, cosmetic surgery). Similarly, VEGF-2 polypeptides or polynucleotides can be used to modulate mammalian metabolism that affects catabolism, anabolism, processing, utilization, and energy storage. Polypeptides or polynucleotides of the VEGF-2 can be used to change the mental state or physical state of the mammal by influencing biorhythms, caricadic rhythms, depression (including depressive disorders), tendency to violence, pain tolerance, reproductive abilities (preferentially by activity) similar to Activin or Inhibin), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

VEGF-2 polypeptides or polynucleotides can also be used as a food additive or preservative, such as to increase or decrease storage capacities, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors or other components nutritional Production of Vectors, Host Cells and Proteins The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention and to host cells containing the recombinant vectors, as well as methods for making such vectors and host cells and for use for the production of VEGF-2 polypeptides or peptides by recombinant techniques. The host cells are genetically engineered (translated, transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector can be, for example, in the form of a plasmid, a viral particle or a bacteriophage, and so on. The engineered host cells can be cultured in modified conventional nutrient media as is appropriate to activate the promoters, select the transformants, or amplify the VEGF-2 genes of the invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the skilled artisan. The polynucleotides of the present invention can be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide sequence can be included in any of a variety of expression vectors to express a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, for example, SV40 derivatives; bacterial plasmids; DNA of bacteriophages; yeast plasmids; vectors derived from combinations of plasmids and bacteriophage DNA, viral DNA such as vaccinia, adenovirus, contagious epithelioma virus, and pseudorabies. However, any other plasmid or vector can be used since it is reproducible and viable in the host. The appropriate DNA sequence can be inserted into the vector by a variety of methods. In general, the DNA sequence is inserted into the appropriate restriction endonuclease site (s) by methods known in the art. Such procedures and others are considered to be within the scope of those skilled in the art. The DNA sequence in the expression vector is operably linked to the appropriate expression control sequence (s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: promoter LTR or SV40, the c or trp of E. coli , the lambda PL promoter of bacteriophages and other known promoters to control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for the initiation of translation and a transcription terminator. The vector may also include the appropriate sequences for the amplification of expression. In addition, expression vectors preferably contain at least one selectable marker gene to provide a phenotypic quality for the selection of transformed host cells. Such markers include resistance to dihydroxaloate reductase (DHFR) or neomycin for the culture of eukaryotic cells, and resistance to tetracycline or ampicillin for E culture. coli and other bacteria. The vector containing the appropriate DNA sequence as described hereinabove, as well as an appropriate promoter or control sequence, can be employed to transform an appropriate host to allow the host to express the protein. Representative examples of appropriate hosts, include but are not limited to: bacterial cells, such as E. col i, Salmonella typhimuri um and Streptomyces; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera S / 9; animal cells such as CHO; COS, and melanoma Bowes; and plant cells. The selection of an appropriate host is considered to be within the reach of those skilled in the art from the teachings herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences described above. The constructs comprise a vector, such as a plasmid or viral vector, in which a sequence of the invention has been inserted, in a forward or inverse orientation. In a preferred aspect of this embodiment, the construct further comprises the regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of vectors and suitable promoters are known to those skilled in the art, and are commercially available. The following vectors are provided by way of example - bacterial: pQE70, pQE60, and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan. In addition to the use of expression vectors in the practice of the present invention, the present invention also includes the novel expression vectors comprising operator elements and promoters operably linked to the nucleotide sequences encoding a protein of interest. An example of such vector is pHE4a which is described in detail below.

As summarized in Figures 28 and 29, the components of the vector pHE4a (SEQ ID NO: 16) include: 1) a neomycin phosphotransferase gene as a selection marker, 2) a duplication origin of E. coli, 3) a T5 promoter sequence of bacteriophages, 4) two operator sequences of the c, 5) a Shine-Delgarno sequence, 6) the repressor gene of the lactose operon (laclg) and 7) a linker region of the cloning site multiple. The duplication origin (oriC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter sequence and the operator sequences were synthetically made. The synthetic production of the nucleic acid sequences is well known in the art. CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East Meadow Circle, Palo Alto, CA 94303. The vector pHE4a was deposited with the ATCC on February 25, 1998, and given the accession number 209645. A nucleotide sequence encoding VEGF-2 (SEQ ID NO: 1), is operably linked to the promoter and pHE4a operator by restricting the vector with Ndel and either Xbal, BamHI, Xhol, or Asp718, and isolating the fragment further large (the region of the multiple cloning site is approximately 310 nucleotides) in a gel. The nucleotide sequence encoding VEGF-2 (SEQ ID NO: 1) having the appropriate restriction sites is generated, for example, according to the PCR protocol described in Example 1, using PCR primers which have restriction sites for Ndel (as the 5 'primer) and either Xbal, BamHI, Xhol, or Asp718 (as the 3' primer) - The PCR insert is a gel purified and restricted with compatible enzymes. The insert and the vector are ligated according to normal protocols. As noted above, vector pHE4a contains a laclg gel. The laclg is an allele of the lacl gene which confers a strict regulation of the operator the c. Amann, E. et al., Gene 69: 301-315 (1988); Stark, M., Gene 52: 255-267 (1987). The laclg gene encodes a repressor protein which binds the operator sequences to c and blocks the transcription of the downstream (ie, 3 ') sequences. However, the product of the lacg gene is dissociated from the operator lac in the presence of either lactose or certain lactose analogs, eg, B-D-isopropyl thiogalactopyranoside (IPTG). In this manner, VEGF-2 is not produced in appreciable amounts in the non-induced host cells containing the vector pHE4a. The induction of these host cells by the addition of an agent such as IPTG, however, results in the expression of the coding sequence of VEGF-2.

The promoter / operator sequences of the vector pHE4a (SEQ ID NO: 17) comprise a T5 promoter of bacteriophages and two lac operator sequences. An operator is located 5 'to the transcriptional start site and the otxo is located 3' to the same site. These operators, when present in combination with a product of the laclq gel, confer a strict repression of the downstream sequences in the absence of an inducer of the operon, eg, IPTG. The expression of the operably linked sequences located downstream of the operators c can be induced by the addition of an operon inducer c, such as IPTG. The binding of a lac inducer to the laclq proteins results in their release from the operator sequences c and the initiation of transcription of the operably linked sequences. The regulation of the operon c of gene expression is observed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807. The pHE4 series of the vectors contain all the components of the pHE4a vector except for the coding sequence of VEGF-2. The characteristics of the pHE4 vectors include the synthetic, optimized bacteriophage T5 promoter, the c operator and the Shine-Delagarno sequences. In addition, these sequences are also optimally separated so that the expression of an inserted gene can be strictly regulated and a high level of expression occurs on induction. Among the bacterial promoters, suitable compounds for use in the production of proteins of the present invention include the lacl and 2acZ promoters of E. coli, the T3 and T7 promoters, the gpt promoter, the PR and PL lambda promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate recent promoter, the HSV thymidine kinase promoter, the SV40 first and subsequent promoters, the retroviral LTR promoters, such as those of the Rous Sarcoma Virus (RSV) and the metallothionein promoters, such as the mouse metallothionein-I promoter. The pHE4a vector also contains a 5 'Shine-Delgarno sequence at the AUG initiation codon. The Shine-Delgarno sequences are short sequences generally located approximately 10 nucleotides upstream (ie, 5 ') of the AUG initiation codon. These sequences essentially direct the prokaryotic ribosomes to the AUG initiation codon.

In this manner, the present invention is also directed to the expression vector useful for the production of the proteins of the present invention. This aspect of the invention is exemplified by the vector pHE4a (SEQ ID NO: 16). Promoter regions can be selected from any desired gene that uses CAT vectors (chloramphenicol transferase) or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Bacterial promoters, named, particular include cl, cZ, T3, T7, gpt, lambda PR, P and trp. Eukaryotic promoters include the first CMV intermediate, HSN thymidine kinase, SV40 first or later, retrovirus LTRs and mouse metallothionein-I. The selection of the appropriate vector and promoter is well within the level of one of ordinary skill in the art. In a further embodiment, the present invention relates to host cells containing the construction described above. The host cell can be a larger eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The introduction of the construction into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran-mediated transfection, electroporation, transduction, infection or other methods (Davis, L., et al., Basic Methods in Molecular Biology (1985)). Constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be produced synthetically by conventional peptide synthesizers. Fully developed proteins can be expressed in mammalian cells, yeast, bacteria or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using the RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Mol ecular Cloning: A Labora tory Manua l, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), the description of which is incorporated by reference by this act.

The transcription of a DNA encoding the polypeptides of the present invention by the higher eukaryotes is increased by inserting an enhancer sequence into the vector. Augmentators are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the last side of the duplication origin (bp 100 to 200), a first cytomegalovirus promoter enhancer, a polyoma enhancer on the last side of the duplication origin, and adenovirus enhancers. In general, recombinant expression vectors will include duplication origins and selectable markers that allow the transformation of the host cell, eg, the ampicillin resistance gene of the TRP1 gene of E. col i and S. cerevisia, and a promoter derived from a highly expressed gene to direct the transcription of a downstream structural sequence. Such promoters can be derivatives of the operons encoding the glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), factor a, acid phosphotase or heat shock proteins, among others. The heterologous structural sequence is coupled in the appropriate phase with the translation initiation and termination sequences, and preferably, a leader sequence capable of directing the secretion of the translated protein into the periplasmic space or the extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that imparts the desired characteristics, eg, stabilization or simplified purification of the recombinant product, expressed. Expression vectors useful for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with the appropriate translation initiation and termination signals in the operable reading phase with a functional promoter. The vector will comprise one or more selectable, phenotypic markers and a duplication origin to ensure maintenance of the vector and, if desired, to provide amplification within the host. Prokaryotic hosts, suitable for transformation include E. col i, Ba ci ll us subti l is, Salmonella typhimuri um and various species within the genus Pseudomonas s, Streptomyces, and Staphylococcus, although others may also be used as a selection subject.

As a representative but not limiting example, expression vectors useful for bacterial use may comprise a selectable marker and a bacterial origin of duplication derived from commercially available plasmids comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017). These commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wl, USA). These "backbone" sections of pBR322 are combined with an appropriate promoter and the structural sequence to be expressed. After transformation of a suitable host strain and development of the host strain to an appropriate cell density, the selected promoter is activated by an appropriate means (eg, change of temperature or chemical induction) and the cells are cultured for an additional period. The cells are typically collected by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. The microbial cells employed in the expression of proteins can be disrupted by any convenient method, well known to those skilled in the art, including the oscillation between freeze-thaw, disruption with high frequency sound waves, mechanical disruption, or use of the cell lysis agents. The various mammalian cell culture systems can also be used to express a recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the cell lines C127, 3T3, CHO, HeLa and BHk. Mammalian expression vectors will comprise a duplication origin, a suitable promoter and an enhancer, and also some necessary ribosine binding site, polyadenylation site, donor and binding acceptor sites, transcriptional termination sequences, and non-transcribed sequences of 5 'flanking. DNA sequences derived from the SV40 viral genome, for example, SV40 origin sites, first promoter, enhancer, linker and polyadenylation can be used to provide the required non-transcribed genetic elements. In addition to encompassing the host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary and immortalized host cells of vertebrate origin, particularly of mammalian origin, that have been designed to suppress or replace the genetic material. , endogenous (eg, a VEGF-2 sequence), and / or to include genetic material (eg heterologous promoters) that is operably associated with the sequence of VEGF-2 of the invention and that activates, alters , and / or amplifies the endogenous VEGF-2 polynucleotides. For example, techniques known in the art can be used to operably associate the heterologous control regions and the endogenous polynucleotide sequences (e.g., encoding VEGF-2) by means of homologous recombination (see, for example. , U.S. Patent No. 5,641,670, filed June 24, 1997, International Publication No. WO 96/29411, published September 26, 1996, International Publication No. WO 94/12650, published on August 4, 1994; Koller et al., Proc. Nal t.Acid. Sci. USA 85: 8932-8935 (1989); and Zijlstra et al., Na ture 342: 435-438 (1989), the descriptions of each of which they are incorporated by reference in their entirety).

The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human-derived cell), or a lower eukaryotic cell, such as a yeast cell or the host cell can be a prokaryotic cell, such as a bacterial cell. The host strain can be selected such that it modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific, desired manner. The expression of certain promoters can be raised in the presence of certain inducers; in this way the expression of the genetically engineered polypeptide can be controlled. In addition, different host cells have specific characteristics and mechanisms for the translation and post-translational process and the modification (eg, glycosylation, phosphorylation, cleavage) of proteins. Appropriate cell lines can be selected to ensure the desired modifications and processing of the expressed protein. Therefore, the polypeptides can be recovered and purified from recombinant cell cultures by the methods used up to now, which include the precipitation of ammonium sulfate or ethanol, acid extraction, anionic or cation exchange chromatography, chromatography of phosphocellulose, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.1-5 mM) of calcium ions present during purification (Price et al., J. Biol. Chem. 244: 911 (1969)). Protein duplication steps can be used, as necessary, to complete the fully developed protein configuration. Finally, high performance liquid chromatography (CLAP) can be used for the final purification steps. The polypeptides of the present invention can be a naturally purified product, or a product of synthetic, chemical processes, or can be produced by the recombinant techniques of a prokaryotic or eukaryotic host (e.g., by bacteria cells, yeast, higher plants , insects and mammals in the crop). Depending on the host employed in a recombinant production method, the polypeptides of the present invention may be glycosylated with the carbohydrates of the mammal or other eukaryotes or may not be glycosylated. The polypeptides of the invention may also include an initial methionine amino acid residue. In addition, the polypeptides of the invention can be chemically synthesized using techniques known in the art (for example, see Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman &Co., NY, and Hunkapiller, M., et al. , 1984, Nature 310: 105-111). For example, a peptide corresponding to a fragment of the VEGF-2 polypeptides of the invention can be synthesized by the use of a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition in the polynucleotide sequence of VEGF-2. Non-classical amino acids include, but are not limited to, the D isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulin, 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 amino acid analogs in general. In addition, the amino acid can be D (dextrorotatory) or L (levorotary). The invention encompasses VEGF-2 polypeptides which are differentially modified during or after translation, eg, by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, binding to a molecule of antibodies or other cellular ligand, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, chemical, specific cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formulation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc. Additional post-translation modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, N-terminal or C-terminal processing), the attachment of chemical portions to the main chain of amino acids, chemical modifications of the N-linked or O-linked carbohydrate chains, and the addition or deletion of an N-terminal methionine residue as a result of the expression of prokaryotic, host cells. The polypeptides can also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity tag to allow suppression and isolation of the protein. Chemically modified derivatives of VEGF-2 are also provided by the invention, which may provide additional advantages such as increased solubility, stability and time of circulation of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical portions for derivatization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol and the like. The polypeptides can be modified at random positions within the molecule, or at predetermined positions within the molecule and can include one, two, three or more chemical moieties, joined.

The polymer can be of any molecular weight, and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about" indicates that in the polyethylene glycol preparations, some molecules will weigh more, some less, than the molecular weight set) for the ease in handling and preparation. Other sizes can be used, depending on the therapeutic profile, desired (for example, the duration of the desired sustained release, the effects, if they exist on the biological activity, the ease of handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol for a therapeutic protein or an analogue). The polyethylene glycol molecules (or other chemical moieties) must be bound to the protein with consideration of the effects on the functional or antigenic domains of the protein. A number of joining methods are available to those skilled in the art, for example, European Patent No. 0 401 384, incorporated herein by reference (coupling of PEG to G-CSF), see also Malik et al., Exp. Hematol. 20: 1028-1035 (1992) (report of the pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently linked through the amino acid residues by means of a reactive group, such as a free amino or carboxyl group. The reactive groups are those to which a polyethylene glycol molecule can be attached. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having the free carboxyl group may include aspartic acid residues, glutamido acid residues and the C-terminal amino acid residue. The sulfhydryl groups can also be used as a reactive group for the attachment of the polyethylene glycol molecules. Binding on an amino group is preferred for therapeutic purposes, such as binding at the N-terminus or a group of lysine. One can specifically desire the chemically modified proteins at the N-terminus. The use of polyethylene glycol as an illustration of the present composition, one can select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of the polyethylene glycol molecules to the protein (or peptide) molecules in the reaction mixture, the type of pegylation reaction to be performed, and the method for obtaining the pegylated protein at the N-terminus, selected. The method for obtaining the pegylated preparation at the N-terminus (ie, the separation of this portion from other mono-pegylated portions if necessary) can be by purification of the pegylated material at the N-terminus of a population of pegylated protein molecules. The chemically modified selective proteins in the modification of the N-terminus can be made by reductive alkylation which exploits the differential reactivity of different types of primary amino groups (lysine against the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, the substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing a polymer is achieved. The VEGF-2 polypeptides of the invention can be in 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 invention, their preparation and compositions (preferably pharmaceutical compositions) containing them. In specific embodiments, the polypeptides of the invention are the monomers, dimers, trimers or tetramers. In the additional embodiments, the multimers of the invention are at least dimers, at least trimer or at least tetramer. The multimers encompassed by the invention can be homomers or heteromers. As used herein, the term "homomer" refers to a multimer containing only VEGF-2 polypeptides of the invention (including VEGF-2 fragments, variants, binding variants, and fusion proteins, as described at the moment) . These homomers may contain the VEGF-2 polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only the VEGF-2 polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing the VEGF-2 polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing VEGF-2 polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing VEGF-2 polypeptides having identical and / or different amino acid sequences). In the further embodiments, the homomeric multimer of the invention is at least one homodimer, at least one homotrimer, or at least one homotetramer. As used herein, the term "heteromer" refers to a multimer containing one or more heterologous polypeptides (ie, different protein polypeptides) in addition to the VEGF-2 polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer or a heterotetramer. In the further embodiments, the homomeric multimer of the invention is at least one homodimer, at least one monotrimer or at least one homotetramer. The multimers of the invention can be the result of hydrophobic, hydrophilic, ionic and / or colvalent associations and / or can be directly linked, for example, by the formation of liposomes. Thus, in one embodiment, the multipliers of the invention, such as, for example, homodimers or homotrimers, are formed when the polypeptides of the invention make contact with one another in the solution. In another embodiment, the heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when the polypeptides of the invention make contact with the antibodies of the polypeptides of the invention (including the antibodies to the heterologous polypeptide sequence). a fusion protein of the invention) in the solution. In other embodiments, the multimers of the invention are formed by covalent associations with and / or between the VEGF-2 polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (for example, that cited in SEQ ID NO: 2, or contained in the polypeptide encoded by the deposited clone). In one example, the covalent associations are crosslinked between the cysteine residues located within the polypeptide sequences which interact in the native polypeptide (i.e., occur naturally). In another example, covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of VEGF-2. In one example, the colvalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, for example, U.S. Patent Number 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a VEGF-2-Fc fusion protein of the invention (as described herein). In another specific example, the covalent associations of the fusion proteins of the invention are between the heterologous polypeptide sequence of another ligand / receptor member of the TNF family that is capable of forming covalently associated multimers, such as, for example, osteoprotegerin (see, for example, International Publication No. Wo 98/49305, the contents of which are incorporated herein by reference in their entirety). The multimers of the invention can be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention can be chemically crosslinked using the linker molecules and linker length optimization techniques known in the art (see, for example, US Patent Number 5,478,925, which - is incorporated herein by reference in its entirety). Additionally, the multimers of the invention can be generated using techniques known in the art to form one or more inter-molecule cross-linkers between the cysteine residues located within the sequence of the desired polypeptides to be contained in the multimer (see, for example , U.S. Patent No. 5,478,925, which is incorporated herein by reference in its entirety). In addition, the polypeptides of the invention can be customarily modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art can be applied to generate the generated multimers containing one or more of these modified polypeptides (see, for example, U.S. Patent Number 5,478,925, which is incorporated herein by reference in its entirety). Additionally, techniques known in the art can be applied to generate liposomes containing the desired polypeptide components to be contained in the multimer of the invention (see, for example, US Patent Number 5,478,925, which is incorporated herein by reference in US Pat. its entirety). Alternatively, the multimers of the invention can be generated using genetic design techniques known in the art. In one embodiment, the polypeptides contained in the multimers of the invention are recombinantly produced using the fusion protein technology described herein or otherwise known in the art (see, e.g., European Patent Number 5,478,925, which is incorporated in the present by reference in its entirety). In a specific embodiment, the polynucleotides encoding a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the product translated from the polypeptide in the reverse orientation of the original C-terminus to the N-terminus (lack of leader sequence) (see, for example, U.S. Patent Number 5,478,925, which is incorporated herein by reference in its entirety). In another embodiment, the recombinant techniques described herein or otherwise known in the art are applied to generate the recombinant polypeptides of the invention which contain a transmembrane domain (or a hydrophobic or signal peptide) and which can be incorporated by liposome membrane reconstitution techniques (see, for example, U.S. Patent Number 5,478,925, which is incorporated herein by reference in its entirety).

Therapeutic Uses The VEGF-2 polypeptide of the present invention is a potent mitogen for endothelial, vascular and lymphatic cells. As shown in Figures 12 and 13, the VEGF-2 polypeptide of SEQ ID NO: 2, minus the initial 46 amino acids, is a potent mitogen for vascular endothelial cells and stimulates their growth and proliferation. The results of a Northern blot analysis performed for the nucleic acid sequence of VEGF-2 encoding this polypeptide wherein 20 mg of RNA from various human tissues were tested with 32p-VEGF-2, illustrate that this protein It is actively expressed in the heart and lungs which is additional evidence of mitogenic activity. Accordingly, VEGF-2, or biologically active portions thereof, can be used to treat vascular trauma by promoting angiogenesis. For example, to stimulate the growth of transplanted tissue where coronary artery bypass surgery is performed. VEGF-2, or biologically active portions thereof, can also be used to promote wound healing, particularly to revascularize damaged tissues or to stimulate collateral blood flow during ischemia and where new capillary angiogenesis is desired. VEGF-2, or biologically active portions thereof, can be used to treat full thickness wounds such as dermal ulcers, including pressure sores, venous ulcers and diabetic ulcers. In addition, VEGF-2, or biologically active portions thereof, can be used to treat full-thickness burns and injuries where a skin graft or flap is used to repair such burns and injuries. VEGF-2, or biologically active portions thereof, can also be employed for use in plastic surgery, for example, for the repair of lacerations, burns or other trauma. In addition, VEGF-2 can be used to promote the healing of wounds and injuries to the eye as well as to treat diseases of the eyes. Along these same lines, VEGF-2, or biologically active portions of it, can also be used to induce the growth of damaged bone, periodontium tissue or ligament. VEGF-2, or biologically active portions thereof, can also be used to regenerate supporting tissues of teeth, including cementum and periodontal ligament, which has been damaged, for example, by periodontal disease or trauma. Since angiogenesis is important in keeping wounds clean and uninfected, VEGF-2, or biologically active portions thereof, can be used in association with surgery and after repair of incisions and cuts. VEGF-2, or biologically active portions thereof, can also be employed for the treatment of abdominal wounds where there is a high risk of infection. VEGF-2, or biologically active portions thereof, can be used for the promotion of endothelialization in vascular graft surgery. In the case of vascular grafts using either transplanted or synthetic material, VEGF-2, or biologically active portions thereof, can be applied to the surface of the graft or at the junction to promote the growth of endothelial, vascular cells. VEGF-2, or biologically active portions of it, can also be used to repair myocardial tissue damage as a result of myocardial infarction. VEGF-2, or biologically active portions thereof, can also be used to repair the vascular, cardiac system after ischemia. VEGF-2, or biologically active portions thereof, can also be employed to treat vascular tissue, damaged as a result of coronary artery disease and vascular, peripheral and CNS disease. VEGF-2, or biologically active portions thereof, it can also be used to coat artificial prostheses or natural organs which must be transplanted in the body to minimize the rejection of the transplanted material and to stimulate the vascularization of the transplanted materials. VEGF-2, or biologically active portions thereof, can also be used for repair of vascular tissue or injuries resulting from trauma, for example, occurring during arteriosclerosis and later requiring balloon angioplasty where the vascular tissues are located. damaged VEGF-2, or biologically active portions thereof, can also be used to treat arterial, peripheral disease. Accordingly, in a further aspect, a method is provided for using the VEGF-2 polypeptides to treat arterial, peripheral disease. Preferably, a VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating arterial, peripheral disease. Suitable dosages, formulations and administration routes are described below. VEGF-2, or biologically active portions thereof, can also promote endothelial function of lymphatic tissues and vessels, such as to treat the loss of lymphatic vessels, occlusions of lymphatic vessels, and lymphangiomas. VEGF-2 can also be used to stimulate lymphocyte production. VEGF-2, or biologically active portions thereof, can also be used to treat hemangioma in newborns. Accordingly, in a further aspect, a process for using the VEGF-2 polypeptides to treat hemangioma in newborns is provided. Preferably, a VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating the hemangioma in newborns. The appropriate dosage, formulations and administration routes are described below. VEGF-2, or biologically active portions thereof, can also be used to prevent or treat retinal, abnormal development in newborn, premature infants. Accordingly, in a further aspect, a process is provided for using the VEGF-2 polypeptides to treat retinal, abnormal development in newborns, premature infants. Preferably, the VEGF-2 polypeptide is administered to an individual for the purpose of relieving or treating retinal, abnormal development in newborns, premature infants. Suitable dosages, formulations and administration routes are described below. VEGF-2, or biologically active portions thereof, can be used to treat primary (idiopathic) lymphademas, including Milroy's disease and Lymphedema praecox. Accordingly, in a further aspect, there is provided a process for using the VEGF-2 polypeptides to treat primary (idiopathic) lymphademas, including Milroy's disease and Lymphedema praecox. Preferably, a VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating primary (idiopathic) lymphademas, including Milroy's disease and Lymphedema praecox. VEGF-2, or biologically active portions thereof, can also be used to treat edema as well as to affect blood pressure in an animal. Suitable dosages, formulations and administration routes are described below. VEGF-2, or biologically active portions of it, can also be used to treat suc- table (obstructive) lifetimes including those resulting from (i) removal of nodes and vessels, (ii) radiotherapy and surgery in the treatment of cancer, and (iii) trauma and infection. Accordingly, in a further aspect, a process is provided for using the VEGF-2 polypeptides to treat secondary (obstructive) lifetimes including those that result from (i) the removal of lymph nodes and vessels, (ii) radiotherapy and surgery in the treatment of cancer, and (iii) trauma and infection. Preferably, a VEGF-2 polypeptide is administered to an individual for the purpose of lifetimes (lifetimes) secondary (obstructive) including those resulting from (i) the removal of lymph nodes and vessels, (ii) radiotherapy and surgery in the treatment of cancer, and (iii) trauma and infection. Suitable dosages, formulations and administration routes are described below. VEGF-2, or biologically active portions thereof, can also be used to treat Kaposi's sarcoma. Accordingly, in a further aspect, a process for using the VEGF-2 polypeptides to treat Kaposi's Sarcoma is provided. Preferably, a VEGF-2 polypeptide is administered to an individual for the purpose of alleviating or treating Kaposi's Sarcoma. The doses, foxmulations and suitable administration routes are described below. Antagonists of VEGF-2 can be used to treat cancer by inhibiting the angiogenesis necessary to support cancer and tumor growth.

Cardiovascular Disorders The present inventors have shown that the VEGF-2 stimulates the growth of endothelial, vascular cells, stimulates the migration of endothelial cells, stimulates angiogenesis in the CAM assay, decreases blood pressure in spontaneously hypertensive rats, and increases blood flow to the ischemic limbs in rabbits. Accordingly, VEGF-2 encoding polypeptides or nucleotides can be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia. Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar syndrome. Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, intercrossed or matted heart, dextrocardia, patent duct arteriosis, Ebstein anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot , transposition of large vessels, double exit right ventricle, tricuspid atresia, persistent truncus arteriosus, and defects of the heart septum, such as aortopulmonary septal defect, endocardic relief defects, Lutembacher syndrome, Fallot trilogy, defects of the ventricular heart. Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), aneurysm of the heart, cardiac arrest, congestive heart failure, congestive cardiomyopathy , paroxysmal dyspnea, cardiac edema, hypertrophy of the heart, congestive cardiomyopathy, left ventricular hypertrophy, hypertrophy of the right ventricle, rupture of the heart after infarction, rupture of the ventricular septum, diseases of the heart valve, myocardial diseases, myocardial ischemia, effusion pericardial, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular complications of pregnancy, Scimitar syndrome, cardiovascular syphilis r and cardiovascular tuberculosis. Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter or flutter, bradycardia, extrasystole, Adams-Stokes syndrome, branch block, sinoatrial block, long QT syndrome, parasyst, Lown-Ganong-Levine syndrome, pre-syndrome. excitement of the Mahaim type, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardia and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic union tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de pointes, and ventricular tachycardia. Heart valve disease includes aortic valve insufficiency, aortic valve stenosis, heart murmurs, prolapse of the aortic valve, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, stenosis of the mitral valve, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency and tricuspid valve stenosis. Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardiac fibroelastosis, endomyocardial fibrosis, Keaxns syndrome, myocardial reperfusion injury, and myocarditis. Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial shock or impact. Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau disease, Klippel-Trenaunay-Weber syndrome, Sturge-Weber syndrome, angioneurotic edema, aortic diseases, Takayasu arteritis, aortitis , Leriche syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolism, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis , pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorthagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis and insufficiency ia venous. The aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, fractured anaerisms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart anaerisms, iliac aneurysms. Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya's disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include diseases of the carotid artery, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arterioclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, breast thrombosis, Wallenberg, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavicular theft syndrome, periventricular leukomalacia, vascular headache, group headache, migraine and vertebrobasilar insufficiency. The embolisms include gas embolisms, embolisms of amniotic fluid, cholesterol embolisms, blue foot syndrome, fatty emboli, pulmonary emboli and thromboembolisms. Thrombolysis includes coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, and thrombophlebitis. Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injury, and ischemia of peripheral limbs. Vasculitis includes aortitis, arteritis, Behcet's syndrome, Churg-Strauss syndrome, lymph node syndrome. mucocutaneous, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis and Wegener's granulomatosis. VEGF-2 polypeptides or polynucleotides are especially effective for the treatment of critical limb ischaemia and coronary artery disease. As shown in Example 18, administration of the polynucleotides and VEGF-2 polypeptides to a rabbit hindlimb with experimentally induced ischemia restored the blood pressure, blood flow, angiographic record, and capillary density ratio. The VEGF-2 polypeptides can be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators. , gels foam sponge deposits, other commercially available reservoir materials, osmotic pumps, solid pharmaceutical, oral or suppository formulations, decantation or topical applications during surgery, aerosol delivery. These methods are known in the art. The VEGF-2 polypeptides can be administered as part of a pharmaceutical composition, described in more detail below. The delivery methods of the VEGF-2 polynucleotides are described in more detail below.

Methods of Gene Therapy Another aspect of the present invention is for gene therapy methods to treat disorders, diseases and conditions. Gene therapy methods refers to the introduction of nucleic acid sequences (DNA, RNA and antisense DNA or RNA) into an animal to achieve expression of the VEGF-2 polypeptide of the present invention. This method requires a polynucleotide which encodes a VEGF-2 polypeptide operably linked to a promoter and any other genetic element necessary for expression of the polypeptide by target or target tissue. This gene therapy and delivery techniques are known in the art, see, for example, WO 90/11092, which is incorporated herein by reference. Thus, for example, the cells of a patient can be designed with a polynucleotide (DNA or RNA) comprising a promoter operably linked to an ex vivo VEGF-2 polynucleotide, with the designed cells which are then provided to a patient to be treated with the polypeptide. These methods are well known in the art. For example, see Belldegrun, A., and collaborators, J. Na ti. Cancer Ins t. 85: 201-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunol. Ogy 253: 4604-4615 (1994); Kaido, T., and collaborators, In t. J. Cancer 50: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., and collaborators, 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)), which are incorporated herein by reference. In one embodiment, the cells, which are designed, are arterial cells. Arterial cells can be reintroduced into the patient through direct injection into the artery, the tissues that surround the artery, or through a catheter injection. As discussed in more detail below, the VEGF-2 polynucleotide constructs can be delivered by any method that delivers the injectable materials to the cells of an animal, such as, an injection into the interstitial space of the tissues (heart, muscle). , skin, lung, liver, and the like). The polypeptide constructs of VEGF-2 can be delivered in a pharmaceutically acceptable liquid or an aqueous carrier. In one embodiment, the VEGF-2 polynucleotide is delivered as a pure polynucleotide. The term "pure" polynucleotide, DNA or RNA refers to sequences that are free of any delivery vehicle that acts to aid, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, agents of lipofectin or precipitation and the like. However, VEGF-2 polynucleotides can also be delivered in liposome formulations and lipofectin formulations and the like can be prepared by methods well known to those skilled in the art. These methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466 and 5,580,859, which are incorporated herein by reference. The vector constructs of the VEGF-2 polynucleotide used in the gene therapy method are preferably constructs that will not integrate into the host genome nor contain the sequences that allow for duplication. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFI / V5, pcDNA3.1, and pRc / CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan. Any strong promoter, known to those skilled in the art, can be used to boost the expression of VEGF-2 DNA. Suitable promoters include adenoviral promoters, such as the posterior, main, adenoviral promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter, the inducible promoters, such as the MMT promoter, the metallothionein promoter; the heat shock promoters; the albumin promoter; the ApoAI promoter; the human globin promoters; the viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; Retroviral LTRs; the b-actin promoter; and the promoters of human growth hormones. The promoter can also be the native promoter for VEGF-2.

Unlike other gene therapy techniques, a major advantage of introducing the pure nucleic acid sequences into the target cells is the transient nature of the synthesis of the polynucleotide in the cells. Studies have shown that DNA sequences without duplication can be introduced into cells to provide production of the desired polypeptide for periods of up to six months. The construction of the VEGF-2 polynucleotide can be delivered to the interstitial space of tissues within an animal, including the muscle, skin, brain, lung, liver, vessel, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testes, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. The interstitial space of the tissues comprises the mucopolysaccharide matrix, fluid, intercellular, between the reticular fibers of tissues of organs, elastic fibers in the walls of vessels or chambers, fibers of collagen of fibrous tissues, or that same matrix within the connective tissue that sheathes the muscle cells or in the gaps of the bone. This is similarly the space occupied by the plasma of the circulation and the lymphatic fluid of the lymphatic channels. The supply to the interstitial space of muscle tissue is preferred for the reasons discussed later. These can be conveniently supplied by an injection into the tissues comprising these cells. These are preferentially delivered to and expressed in non-dividing, persistent cells which are differentiated, although delivery and expression can be achieved in undifferentiated or less completely differentiated cells, such as, for example, fibroblast stem cells of blood and skin. Muscle cells in vivo are particularly competent in their ability to take and express the polynucleotides. For injection of the pure acid sequence, an amount of effective dose of DNA or RNA will be in the range of about 0.05 mg / kg of body weight to about 50 mg / kg of body weight. Preferably, the dosage will be from about 0.005 mg / kg to about 20 mg / kg and more preferably from about 0.05 mg / kg to about 5 mg / kg. Naturally, as the architect of ordinary experience will appreciate, this dosage will vary according to the tissue site of the injection. The appropriate and effective dose of the nucleic acid sequence can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral injection route in the interstitial space of the tissues. However, other parenteral routes may also be used, such as inhaling an aerosol formulation particularly for delivery to tissues of the lungs or bronchi, throat or mucous membranes of the nose. In addition, DNA constructs of pure VEGF-2 can be delivered to the arteries during angioplasty by the catheter used in the procedure. Pure polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and those commonly referred to as "gene guns". These delivery methods are known in the art. As is evident from Example 18, the nucleic acid sequences of pure VEGF-2 can be administered in vivo which results in the successful expression of the VEGF-2 polypeptide in the femoral arteries of rabbits.

Constructs can also be supplied with delivery vehicles such as viral sequences, viral particles, liposome formulations, lipofectin, precipitation agents, etcetera. These delivery methods are known in the art. In certain embodiments, the polynucleotide constructs of VEGF-2 are made complex in a liposome preparation. Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a sparse charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to measure the intracellular delivery of plasmid DNA (Felgner et al, Proc.Na ti, Acad.Sci, USA (1987) 84: 7413-7416, which is incorporated herein by reference); MRNA (Malone- et al., Proc.Nal t.Accid. Sci. USA (1989) 85: 6077-6081, which is incorporated herein by reference); and purified transcription factors (Des et al., J. Bi ol. Chem. (1990) 255: 10189-10192, which is incorporated herein by reference), in functional form. Cationic liposomes are readily available. For example, [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Na ti Acad. Sci. USA (1987) 84: 7413-7416, which is 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. See, for example, PCT Publication No. WO 90/11092 (which is incorporated herein by reference) for a description of the synthesis of liposomes of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonium). propane). The preparation of DOTMA liposomes is explained in the literature, see, for example, P. Felgner et al., Proc. Na ti. Acad. Sci. USA 84: 1413-7417, which is incorporated herein by reference. Similar methods can be used to prepare liposomes of other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala), or can be easily prepared using readily available materials. These materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphosphatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making the liposomes using these materials are well known in the art. For example, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG / DOPC vesicles can be prepared by drying 50 mg of each DOPG and DOPC under a stream of nitrogen gas in a disrruption flask with high frequency sound waves. The sample is placed under a vacuum pump overnight and hydrated the next day with deionized water. The sample is then disrupted with high frequency sound waves for 2 hours in a capped flask, using a high frequency sound waves switch Heat Systems, model 350, equipped with a concave, inverted probe (bath type) at the maximum setting while the bathroom is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without disruption with high frequency sound waves to produce multilamellar vesicles or by extrusion through nucleoporous membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those skilled in the art. Liposomes can comprise multilamellar vesicles (MLVs), unilamellar vesicles, small (SUVs), or large unilamellar vesicles (LUVs), with SUVs that are the preferred ones The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, for example, Straubinger et al., Methods of Immunology (1983), 101: 512-521, which is incorporated herein by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipids on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by disrupting high-frequency, extended-frequency sound waves of the MLVs to produce a homogenous population of unilamellar liposomes. The material that is trapped is added to a suspension of the preformed MLVs and then disrupted with high frequency sound waves. When liposomes containing cationic lipids are used, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic, buffer solution such as 10 mM Tis / NaCl, disrupted with high frequency sound waves, and then the realized liposomes are mixed directly with the DNA. The liposome and the DNA form form a very stable complex due to the binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. The LUVs are prepared by a number of methods, well known in the art. Commonly used methods include formation of Ca2 + -EDTA chelates (Papahadj opoulos et al., Biochim, Bi ophys, Acta (1975) 394: 483, Wilson et al. Cell (1979) 27:77); ether injection (Deamer, D. and Banham, A., Biochim Biophys. Acta (1976) 443: 629; Ostro et al., Bi ochem. Bi ophys. Res. Commun. (1977) 75: 836; Fraley et al. , Proc. Na ti. Acad. Sci. USA (1979) 75: 3348); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Na ti. Acad. Sci. USA (1979) 75: 145); and reverse phase evaporation (REV) (Fraley et al., J. Biol. Chem. (1980), 255: 10431; Szoka, F. and Papahadj opoulos, D., Proc. Na ti. Sci. USA (1978) 75: 145; Schaefer-Ridder et al., Sci ence (1982) 215: 166), which are incorporated herein by reference. In general, the ratio of DNA to liposomes will be from about 10: 1 to about 1:10. Preferably, the ratio will be from about 5: 1 to about 1: 5. More preferably, the ratio will be from about 3: 1 to about 1: 3, even more preferably, the ratio will be about 1: 1. U.S. Patent No. 5,676,954 (which is incorporated herein by reference) reports on the injection of genetic material, formed in complex with the carriers of cationic liposomes, in mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 4,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no.

WO 94/9469 (which are incorporated herein by reference) provide cationic lipids for use in transfecting DNA in cells and mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055 and international publication no. WO 94/9469 (which are incorporated herein by reference) provide methods for delivering cationic DNA-lipid complexes to mammals. In certain embodiments, cells must be designed, ex vi ve or in vi ve, using a retroviral particle containing RNA which comprises a sequence encoding VEGF-2. Retroviruses from which vectors of retroviral plasmids can be released include, but are not limited to, Moloney Murine Leukemia Virus, vessel necrosis virus, Rous Sarcoma Virus, Harvey Sarcoma Virus, bird leukosis virus, gibbon monkey leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. The retroviral plasmid vector is used to transduce the packaging cell lines to form the producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, cell lines PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE , RCRIPT, GP + E-86, GP + envAml2, and DNA as described in Miller, Human Gene Therapy 2: 5-14 (1990), which is "incorporated herein by reference in its entirety. transducing the packaging cells through any means known in the art These means include, but are not limited to, electroporation, the use of liposomes, and CaP0 precipitation.In an alternative, the retroviral plasmid vector can be encapsulated in a liposome or can be coupled to a lipid, and then administered to a host.The line of producing cells generates infectious retroviral vector particles which include the polynucleotide that encodes VEGF-2 .These retroviral vector particles can then be used , to transduce eukaryotic cells cas, either in 'vi tro or in vivo. The transduced eukaryotic cells will express VEGF-2. In certain embodiments, the cells are designed, ex vivo or in vivo, with the VEGF-2 polynucleotide contained in an adenovirus vector. The adenovixus can be manipulated such that it cohesifies and expresses VEGF-2, and at the same time it is deactivated in terms of its ability to duplicate itself in the Viral, lytic, normal Life cycle. Expression of adenovirus is achieved without integration of viral DNA into the chromosome of host cells, because of this relief is related to insertion mutagenesis. In addition, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schawartz, A. R. et al. (1974) Am. Rev. Respir. Dis. 109: 233-238). Finally, adenovirus-mediated gene transfer has been demonstrated in a number of examples including the transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, MA et al. (1991) Science 252: 431-434). Rosenfeld et al. (1992) Cel l: 143-155). In addition, extensive studies to try to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Na ti. Acad. Scí. EUA 75: 6606). Suitable adenoviral 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 58: 143-155 (1992); Engelhardt et al., Human Gene-t. Ther. 4: 759-769 (1993); Yang and collaborators, Na ture Genet. 7: 362-369 (1994); Wilson et al., Na ture 355: 691-692 (1993); and the North American patent No. ,652,224, which are incorporated herein by reference. For example, Ad2 adenovirus vector is used and can be developed in human 293 cells. These cells contain the El region of the adenovirus and constitutively express Ela and Elb, which complement the defective adenovirus by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenoviruses (eg, Ad3, Ad5 and Ad7) are also useful in the present invention. Preferably, the adenovirus used in the present invention are deficient in duplication. Adenoviruses deficient in duplication require the aid of an helper virus and / or a packaging cell line to form infectious particles. The resulting virus is capable of infecting the cells and can express a polynucleotide of interest which is operably linked to a promoter, for example, the HARP promoter of the present invention, but can not be duplicated in most cells. Adenoviruses deficient in duplication can be deleted in one or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a or Ll up to L5.

In other certain modalities, the cells are designed, ex vivo or in vivo, using an adeno-associated virus (AAV, for its acronym in English). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topi cs in Mi crobiol, Immunol 158: 91 (1992)). This is also one of some viruses that can integrate their DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and integrated, but the space for the exogenous DNA is limited to approximately 4.5 kb. Methods for producing and using such AVVs are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745 and 5,589,377. For example, an AAV vector suitable for use in the present invention will include all sequences necessary for the duplication, encapsulation and integration into the host cells of the DNA. The VEGF-2 polynucleotide construct is inserted into the AAV vector using normal cloning methods, such as those found in Sambrook et al., Mol ecula r Cloning: A Labora tory Manua l, Cold Spring Harbor Press (1989). The vector of the recombinant AAV is then transfected into the packaging cells which are infected with a helper virus, using any normal technique, including lipofection, electroporation, calcium phosphate precipitation, and so on. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the construction of the VEGF-2 polynucleotide. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the construction of the VEGF-2 polynucleotide integrated into their genome, and will express VEGF-2. Another method of gene therapy involves the heterologous control regions, in operable association and the endogenous polynucleotide sequences (e.g. encoding VEGF-2) by means of homologous recombination (see, for example, U.S. Patent No. 5,641,670 , filed June 24, 1997, International Publication No. WO 96/29411, published September 26, 1996, International Publication No. WO 94/12650, published August 4, 1994, Koller et al., Proc. Na ti, Acad. Sci.

USA 85: 8932-8935 (1989); and Zijlstra et al., Na ture 342: 435-438 (1989). This method involves the activation of a gene which is present in the target or target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired. The polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with target or target sequences lateral to the promoter. Suitable promoters are described herein. The target or target sequence is sufficiently complementary for an endogenous sequence to allow homologous recombination of the promoter-target sequence with the endogenous sequence. The target sequence will be sufficiently close to the 5 'end of the desired endogenous VEGF-2 polynucleotide sequence so that the promoter will be operably linked to the endogenous sequence in the homologous recombination. The promoter and target sequences can be amplified using PCR. Preferably, the amplified promoter contains different restriction enzyme sites at the 5 'and 3' ends. Preferably, the 3 'end of the first target sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second target sequence contains the same restriction site at the 3' end. of the amplified promoter. The amplified promoter and the target sequences are summarized and linked together. The construction of the promoter-target sequence is supplied to the cells, either as a pure polynucleotide, or in conjunction with agents that facilitate transfection, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitation agents, etc., described in more detail above. The promoter-target sequence P may be delivered by any method, including direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, and the like. The methods are described in more detail later. The construction of the promoter-target sequence is taken by the cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous VEGF-2 sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence of VEGF-2. The polynucleotides encoding VEGF-2 can be administered together with other polynucleotides that encode other angiogenic proteins. Angiogenic proteins include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, alpha and beta epidermal growth factor, platelet-derived endothelial cell growth factor, growth factor. platelet derivative, alpha tumor necrosis factor, hepatocyte growth factor, insulin-like growth factor, colony stimulation factor, macrophage colony stimulation factor, colony stimulation factor of granulocytes / marcófagos and nitric oxide synthase. Preferably, the polynucleotide encoding VEGF-2 contains a sequence of secretory signals that facilitates the secretion of the protein. Typically, the signal sequence is placed in the coding region of the polynucleotide to be expressed towards or at the 5 'end of the coding region. The signal sequence can be homologous or heterologous to the polynucleotide of interest and can be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence can be chemically synthesized using methods known in the art. Any mode of administration of any of the polynucleotide constructs described above can be used since the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, biolistic injectors, particle accelerators (ie, "gene guns"), gelspuma sponge reservoirs, other commercially available reservoir materials, osmotic pumps (e.g. Alza minipumps), solid, oral or suppository pharmaceutical formulations (tablet or pill) and decanting or topical applications during surgery. For example, direct injection of a precipitated plasmid of pure calcium phosphate into rat liver and rat vessel or a protein-coated plasmid into the portal vein has resulted in the expression of foreign gene genes in liver livers. rat (Kaneda et al., Science 243: 315 (1989)).

A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the artery area. The administration of a composition locally within the area of the arteries refers to the injection of the centimeters of composition and preferably, millimeters into the arteries. Another method of local administration is to contact a construction of the polynucleotide of the present invention in or around the surgical wound. For example, a patient may undergo surgery and the construction of the polynucleotide may be coated on the surface of the tissue within the wound or the construct may be injected into the areas of the tissue within the wound. Therapeutic compositions useful in systemic administration, include the recombinant molecules of the present invention complexed with a target delivery vehicle of the present invention. Adequate delivery vehicles for use with systemic administration comprise liposomes comprising ligands to target or target the vehicle for a particular site. Preferred methods of systemic administration include intravenous injection, aerosol delivery, oral and percutaneous (topical): Intravenous injections can be performed using standard methods in the art. The aerosol delivery can also be performed using standard methods in the art (see, for example, Stribling et al, Proc.Na ti.Acid Sci.U.A. 289: 11277-11281, 1992, which is incorporated herein by reference. reference). Oral delivery can be performed by complexing a construction of the polynucleotide of the present invention with a carrier capable of resisting degradation by digestive enzymes in the intestines of an animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. Topical delivery can be carried out by mixing a construction of the polynucleotide of the present invention with a lipophilic reagent (eg, DMSO) which is capable of passing into the skin. The determination of an effective amount of the substance to be delivered may 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 precise condition required by the animal. treatment and its severity, and the route of administration. The frequency of treatments depends on a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, the dosage number, and the timing of the doses will be determined by the attending physician or veterinarian. The therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cattle, horses and pigs, with humans being particularly preferred.

Nucleic Acid Utilities The nucleic acid sequences of VEGF-2 and the VEGF-2 polypeptides can also be used for in vi tro purposes related to scientific research, DNA synthesis and the manufacture of DNA vectors, and for the production of diagnostic and therapeutic substances to treat a human disease. For example, VEGF-2 can be used for vascular endothelial cell culture, where it is added to the conditional medium at a concentration of 10 pg / ml to 10 ng / ml. Fragments of the full-length VEGF-2 gene can be used as a hybridization test substance for a cDNA library to isolate other genes which have a high high sequence similarity to the gene or similar biological activity. Test substances of this type generally have at least 50 base pairs, although these may have a higher number of bases. The test substance can also be used to identify a cDNA clone corresponding to a full-length transcript and a clone or genomic clones containing the complete VEGF-2 gene including the regulatory regions and promoters, exons and introns. An example of a selection comprises isolating the coding region of the VEGF-2 gene by using the known DNA sequence to synthesize an oligonucleotide test substance. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to select a human cDNA library, genomic DNA or mRNA to determine which members of the library hybridize the test substance. This invention provides methods for the identification of VEGF-2 receptors. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, separation by washing with ligands and the FACS classification (Coligan et al., Curren t Protocols in Immun., 2 (2) , Chapter 5, (1991)). Preferably, expression cloning is employed wherein the polyadenylated RNA is prepared from a cell responsive to VEGF-2, and a cDNA library created from this RNA is divided into clusters and used to transfect COS cells. other cells that are not responsive to VEGF-2. Transfected cells which are cultured on glass slides are exposed to labeled VEGF-2. VEGF-2 can be labeled by a variety of means including iodination or the inclusion of a recognition site for a site-specific protein kinase. After fixation and incubation, the slides are subjected to autoradiographic analyzes. Positive clusters are identified and sub-clusters are prepared and transferred using an iterative sub-clustering and reselection process, eventually producing an individual clone that encodes the putative receptor. As an alternative approach for the identification of the receptor, the labeled VEGF-2 can be linked by photoaffinity with the cell membrane or extract preparations expressing the receptor molecule. The crosslinked material is resolved by the PAGE and exposed to a X-ray film. The labeled complex containing the VEGF-2 is then excited, resolved into the peptide fragments and subjected to the array of protein microsequences. The amino acid sequence obtained from the array of microsequences would be used to design a set of degenerate oligonucleotide test substances to select a cDNA library to identify the gene encoding the putative receptor.VEGF-2 Agonists and Antagonists This invention also relates to a method for selecting compounds to identify those that are agonists or antagonists of VEGF-2. An 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 the A-comitogen. The endothelial cells are obtained and cultured in 96-well flat bottom culture plates. (Costar, Cambridge, MA), in a reaction mixture supplemented with Con-A (Calbiochem, La Jolla, CA). The Con-A, the polypeptides of the present invention and the compound to be selected are added. After incubation at 37 ° C, the cultures were boosted with 1 Fci of 3 [H] thymidine (5 Ci / mmol, 1 Ci = 37 BGq, NEN) for a sufficient time to incorporate the 3 [H] and collected on filters of fiberglass (Cambridge Technology, Waertown, MA). The incorporation of 3 [H] -i idina average (cpm) of the cultures in triplicate is determined using a liquid scintillation counter (Beckman Instruments, Irvine, CA). The incorporation of 3 [H] thymidine, compared to a control assay where the compound is excluded, indicates the stimulation of endothelial cell proliferation. To test the antagonists, the assay described above is performed and the ability of the compound to inhibit the incorporation of 3 [H] thymidine in the presence of VEGF-2 indicates that the compound is an antagonist to VEGF-2. Alternatively, VEGF-2 antagonists can be detected by combining VEGF-2 and a potential antagonist with VEGF-2 receptors bound to the membrane or recombinant receptors under conditions appropriate for a competitive inhibition assay. VEGF-2 can be labeled, such as by radioactivity, such that the number of VEGF-2 molecules bound to the receptor can determine the effectiveness of the potential antagonist. Alternatively, the response of a second messenger system, known after the interaction of VEGF-2 and the receptor, would be measured and compared in the presence or absence of the compound. These second messenger systems include, but are not limited to, guanylate cyclase cAMP, ion channels or phosphoinositide hydrolysis. In another method, a mammalian cell or a membrane preparation expressing the VEGF-2 receptor is incubated with the labeled VEGF-2 in the presence of the compound. The ability of the compound to increase or block this interaction could then be measured. Potential antagonists of VEGF-2 include an antibody, or in some cases, an oligonucleotide, which binds to the polypeptide and effectively eliminates the function of VEGF-2. Alternatively, a potential antagonist may be a closely related protein which binds to the VEGF-2 receptors, however, these are inactive forms of the polypeptide and thereby impede the action of VEGF-2. Examples of these antagonists include a dominant, negative mutation of the VEGF-2 polypeptide, for example, a strand of the hetero-dimeric form of VEGF-2 may be dominant and may be mutated such that biological activity is not retained. An example of a dominant, negative mutation includes truncated versions of a dimeric VEGF-2 which is capable of interacting with another dimer to form VEGF-2 of the non-cultured type, however, the resulting homo-dimer is inactive and fails in exhibit the characteristic activity of VEGF. Another potential VEGF-2 antagonist is an antisense or complementary sequence construct prepared using antisense technology. Antisense technology can be used to control the expression of genes through triple helix formation or DNA or antisense RNA, both of which are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which encodes the fully developed polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucí Acids Res. 5: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., 252: 1360 (1991)), because of this transcription and production of VEGF-2 is impeded. The antisense RNA oligonucleotide hybridizes to the AR? in vivo and blocks the translation of the AR [mu] m molecule into the VEGF-2 polypeptide (Antisense-Okano, J. Neurochem, 55: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expresssion, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to the cells such that the AR? or the AD? Antisense can be expressed in vivo to inhibit the production of VEGF-2. Potential antagonists of VEGF-2 also include small molecules which bind to and occupy the active site of the polypeptide due to which the catalytic site inaccessible to the substrate is marked such that normal, biological activity is impeded. Examples of small molecules include, but are not limited to, small peptides or molecules similar to the peptide. Antagonists can be used to limit the angiogenesis necessary for the metastasis of solid tumors. The identification of VEGF-2 can be used for the generation of certain inhibitors of vascular endothelial growth factor. Since angiogenesis and neovascularization are essential steps in the growth of solid tumors, the inhibition of the angiogenic activity of vascular endothelial growth factor is very useful to prevent further growth, retardation or even the return of solid tumors. Although the level of expression of VEGF-2 is extremely low in normal tissues including the breast, it can be found expressed at moderate levels in at least two breast tumor cell lines that are derived from malignant tumors. Therefore, it is possible that VEGF-2 is involved in angiogenesis and tumor growth. Gliomas are also a type of neoplasm that can be treated with the antagonists of the present invention. Antagonists can also be used to treat chronic inflammation caused by increased vascular permeability. In addition to these disorders, antagonists can also be used to treat retinopathy associated with diabetes, rheumatoid arthritis and psoriasis.

The antagonists can be employed in a composition with a pharmaceutically acceptable carrier, or example, as described hereinafter.

Pharmaceutical Compositions Polypeptides, polynucleotides and agonists and antagonists of VEGF-2 can be used in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide or agonist or antagonist, and a pharmaceutically acceptable carrier or excipient. This carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must adapt to the mode of administration. The invention also provides a pharmaceutical package or equipment comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with this (these) recipient (s) may be a notice in written form • by a governmental agency that regulates the manufacture, use or sale of pharmaceutical compositions or biological products, the notice that reflects the approval by the manufacturing agency , use or sale for human administration. In addition, the pharmaceutical compositions can be used in conjunction with other therapeutic compounds. The pharmaceutical compositions can be administered in a convenient manner such as by topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for the treatment and / or prophylaxis of the specific indication. In general, the pharmaceutical compositions are administered in an amount of at least about 10 mg / kg of body weight and in most cases these will be administered in an amount not greater than about 8 mg / kg of body weight per day. In most cases, the dose is from about 10 mg / kg to about 1 mg / kg of body weight per day, taking into account the routes of administration, symptoms, etc. VEGF-2 polypeptides, and agonists or antagonists which are polypeptides, can also be employed according to the present invention by expressing such a polypeptide in vivo, which is often referred to as "gene therapy" described above. . In this way, for example, cells such as bone marrow cells can be designed with a polynucleotide (DNA or RNA) encoding the polypeptide ex vivo, then designed cells were provided to a patient to be treated with the polypeptide. These methods are well known in the art. For example, the cells can be designed by methods known in the art by the use of a retroviral particle containing the RNA encoding the polypeptide of the present invention. Similarly, the cells can be designed in vivo for the expression of a polypeptide in vivo, for example, by methods known in the art. As is known in the art, a producer cell for producing a retroviral particle containing the RNA encoding a polypeptide of the present invention can be administered to a patient to design the cells in vivo and the expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such methods should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for designing the cells may be different from a retroviral particle, for example, an adenovirus, which may be used to design the cells in vivo after combination with a suitable delivery vehicle. Retroviruses from which the retroviral plasmid vectors mentioned hereinabove can be derived include, but are not limited to, Moloney Murine Leukemia Virus, vessel necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, bird leukosis virus, gibbon monkey leukemia virus, human immunodeficiency virus , adenovirus, myeloproliferative sarcoma virus and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus. The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques 7: 980-990 (1989) or any other promoter (eg, cellular promoters such as eukaryotic cell promoters including, but not limited to, histone, pol III and b-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters and parvovirus B19 promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein. The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the posterior, main, adenoviral promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus promoter (RSV), inducible promoters, such as the promoter MMT, the promoter of metallothionein; heat shock promoters; the albumin promoter, the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the herpes simplex thymidine kinase promoter, retroviral LTRs (includes the retroviral LTRs, modified described above); the b-actin promoter; and the promoters of human growth hormone. The promoter may also be the native promoter which controls the gene encoding the polypeptide. The retroviral plasmid vector is used to transduce the packaging cell line to form the producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, cell lines PE501, PA317, y-2, y-AM, PA12, T19-14X, VT-19-17-H2, and CRE , yCRIP, GP + E-86, GP + envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes and the precipitation of CaP04. In an alternative, the retroviral plasmid vector can be encapsulated in a liposome, or can be coupled to a lipid, and then administered to a host. The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence (s) encoding the polypeptides. These retroviral vector particles can then be used to transduce the eukaryotic cells, either in vitro or in vivo. The eukaryotic, transduced cells will express the nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells which can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells and epithelial, bronchial cells.

Diagnostic Assays This invention also relates to the use of the VEGF-2 gene as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the nucleic acid sequences of VEGF-2. Individuals carrying mutations in the VEGF-2 gene can be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis can be obtained from patient cells, such as blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by the use of PCR (Saiki et al., Na ture 324: 163-166 (1986)) before analysis. RNA or cDNA can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding VEGF-2 can be used to identify and analyze mutations of VEGF-2. For example, deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to the radiolabelled VEGF-2 RNA or alternatively, the antisense DNA sequences of the radiolabelled VEGF-2. The perfectly linked sequences can be distinguished from mismatched duplos by the digestion of Rnase A or by the differences in the melting temperatures. The test or genetic analysis based on the differences of the DNA sequences can be achieved by detecting the alteration in the electrophoretic mobility of the DNA fragments in gels with or without denaturing agents. Suppressions and insertions of small sequences can be visualized by high-resolution gel electrophoresis. The DNA fragments of different sequences can be distinguished in the denaturation of formamide gradient gels in which the mobilities of the different DNA fragments are delayed in the gel in different positions according to their specific melting or partial melting temperatures. (see, for example, Myers et al., Science 230: 1242 (1985)). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical division method (eg, Cotton et al., PNAS, USA 85: 4397-4401). (1985)). In this way, the detection of a specific DNA sequence can be achieved by methods such as hybridization, RNase protection, chemical division, ordering of direct DNA sequences or the use of restriction enzymes, (e.g. , Restriction Fragment Length Polymorphisms (RFLP)) and Southern blot analysis of genomic DNA. In addition to the more conventional gel electrophoresis and the ordering of DNA sequences, mutations can also be suppressed by in situ analysis. The present invention also relates to a diagnostic assay for detecting altered levels of the VEGF-2 protein in various tissues since an overexpression of the proteins compared to normal control tissue samples can detect the presence of a disease or susceptibility to a disease, for example, cellular differentiation, abnormal. Assays used to detect VEGF-2 protein levels in a sample derived from a host are well known to those skilled in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, ELISA assays and "sandwich". An ELISA assay (Coligan et al., Curren t Protocols in Immunology 1 (2), Chapter 6, (1991)) comprises initially preparing an antibody specific for the VEGF-2 antigen, preferably a monoclonal antibody. In addition, a reporter antibody is prepared against the monoclonal antibody. A detectable reagent such as radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme is attached to the reporter antibody. A sample is separated from a host and incubated on a solid support, for example, a polystyrene dish, which binds the proteins in the sample. Any of the free protein binding sites in the dish are then covered by incubating with a non-specific protein, such as bovine serum albumin. Then, the monoclonal antibody is incubated at the plate during which the monoclonal antibodies bind to any of the VEGF-2 proteins bound to the polystyrene plate. All unbound monoclonal antibody is washed with the buffer. The reporter antibody bound to horseradish peroxidase is placed in the dish which results in the binding of the reporter antibody to any monoclonal antibody bound to VEGF-2. The unbound report antibody is then washed. The peroxidase substrates are then added to the dish and the amount of color developed in a given period of time is a measure of the amount of the VEGF-2 protein present in a given volume of the patient sample when compared against a curve normal. A competition assay can be employed wherein the specific antibodies for VEGF-2 bind to a solid support. The polypeptides of the present invention are then labeled, for example, by radioactivity, and a sample derived from the host is passed over the solid support and the amount of label detected, for example by liquid scintillation chromatography, can be correlated to an amount of VEGF-2 in the sample.

A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay, VEGF-2 is passed over a solid support and bound to an antibody bound to a solid support. A second antibody then binds to VEGF-2. A third antibody which is labeled and specific for the second antibody is then passed over the solid support and binds to the second antibody and then an amount can be measured.

Chromosome Identification The sequences of the present invention are also valuable for the identification of chromosomes. The sequence is targeted or targeted specifically for and can be hybridized to a particular location on an individual human chromosome. In addition, there is a current need to identify the particular sites on the chromosome. Some reagents that mark chromosomes based on current sequence data (repeat polymorphisms) are currently available to mark the chromosomal location. The location of the DNAs in the chromosomes according to the present invention is an important first step in the correlation of those sequences with the genes associated with the disease.

In summary, the sequences can be located on the chromosomes when preparing the PCR primers (preferably 15-25 bp) of the cDNA. Computer analysis of the cDNA is used to quickly select primers that do not extend over more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for the selection with the PCR of somatic cell hybrids containing the individual human cormosomes. Only those hybrids containing the human gene corresponding to the primer will produce an amplified fragment. The location with the PCR of the somatic cell hybrids is a rapid procedure to assign a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments of the specific chromosomes or clusters of large genomic clones in an analogous manner. Other location strategies that can be used similarly for the location on your chromosome include hybridization in itself, preselection with the chromosomes classified by the flow, labeling and preselection by hybridization to build chromosome-specific cDNA libraries. Fluorescence in si t u hybridization (FISH) from a cDNA clone to a chromosomal metaphase extension can be used to provide a chromosomal location, accurate in one step. This technique can be used with cDNA test substances as short as 50 or 60 base pairs. For a review of this technique, see Verma et al., Human Chromosomes: a Manua l of. Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been located in a chromosomal location, accurate, the physical position of the sequence on the chromosome can be correlated with the genetic location data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through the Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been located in the same chromosomal region are then identified through linkage analysis (common inheritance of physically adjacent genes). Then, if necessary, differences in the cDNA or genomic sequence between affected or unaffected individuals are determined. If a mutation is observed in some or all of the affected individuals but not in some normal individual, then the mutation is likely to be the causative agent of the disease. With the current resolution of the techniques of physical location and genetic location, a cDNA located precisely in a chromosomal region associated with the disease could be one of between 50 and 500 potential genes. (This assumes a resolution of 1 megabase location and one gene per 20 kb). Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from the extensions of the chromosome or detectable using PCR based on that cDNA sequence. Finally, it requires the ordering of complete sequences of genes from different individuals to confirm the presence of a mutation and to distinguish mutations of polymorphisms.

ANTI-SENSE The present invention is further directed to the inhibition of VEGF-2 in vivo by the use of antisense technology. Antisense technology can be used to control the expression of genes through triple-helix formation or DNA or antisense RNA, both of which are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the fully developed nucleotide sequence, which encodes the polypeptide of the present invention, is used to designate an antisense RNA oligonucleotide of 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple-helix - see Lee et al., Nucí Acids Res. 5: 3073 (1979); Cooney et al., Science 242: 456 (1988); and Dervan et al., Science, 251: 1360 (1991), because of this transcription and production of VEGF-2 is impeded. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks the translation of a mRNA molecule in VEGF-2 (antisense-Okano, J. Neurochem, 55: 560 (1991)): Oligoigoxynucl eo tides as An tisense Inhibi tors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Alternatively, the oligonucleotides described above can be delivered to the cells by methods in the art such that the antisense RNA or DNA can be expressed in vi to inhibit the production of VEGF-2 in the manner described above. Therefore, antisense constructs for VEGF-2 can inhibit the angiogenetic activity of VEGF-2 and prevent further growth or even the return of human tumors, since angiogenesis and neovascularization are essential steps in tumor growth. solid These antisense constructs can also be used to treat rheumatoid arthritis, psoriasis, diabetic retinopathy and Kaposi's sarcoma which are all characterized by abnormal angiogenesis.

Portions Carrying Epitopes In another aspect, the invention provides peptides and polypeptides comprising epitope-bearing portions of the polypeptides of the present invention. These epitopes are immunogenic or antigenic epitopes of the polypeptides of the present invention. An "immunogenic epitope" is defined as a part of a protein that produces an antibody response in vivo when the complete polypeptide of the present invention, or a fragment thereof, is the immunogen. On the other hand, a region of a polypeptide, to which an antibody can be attached, is defined as an "antigenic determinant" or "antigenic epitope". The number of immunogenic epitopes in vi of a protein in general is less than the number of antigenic epitopes. See, for example, Geysen et al. (1983) Proc. Nati Acad. Sci. USA 81: 3998-4002. However, antibodies can be made for any antigenic epitope, taking into account whether it is an immunogenic epitope, by using methods such as visual representation of bacteriophages. See, for example, Petersen G. et al. (1995) Mol. Gen. Genet. 249: 245-431. Therefore, both the immunogenic epitopes and the antigenic epitopes are included in the present invention. It is particularly noted that the immunogenic epitopes comprise predicted, critical amino acid residues determined by the Jameson-Wolf analysis. In this manner, additional lateral residues in either the N-terminal, C-terminal or both N-terminal and C-terminal ends can be added to these sequences to generate a polypeptide carrying epitopes of the present invention. Therefore, the immunogenic epitopes can include additional N-terminal or C-terminal amino acid residues. Additional lateral amino acid residues may be contiguous with the N-terminal and / or C-terminal, lateral sequences of the polypeptides of the present invention, the heterologous polypeptide sequences, or may include both contiguous, lateral sequences of the polypeptides of the present invention and heterologous polypeptide sequences. The polypeptides of the present invention comprising immunogenic or antigenic epitopes are at least 7 amino acid residues in length. "At least" means that a polypeptide of the present invention comprising an immunogenic or antigenic epitope can be 7 amino acid residues in length or any number between 7 amino acids and "the number of amino acid residues of the full length polypeptides of the invention Preferred polypeptides comprising immunogenic or antigenic epitopes are at least lateral 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length, however, it is noted that each and every one of the integers between 7 and the number of amino acid residues of the full-length polypeptide are included in the present invention. Epitopes, immunogens, and antigens can be specified by either the number of contiguous amino acid residues, as described above, or they can be further specified by the N-terminal and C positions. -terminals of these fragments in the amino acid sequence of SEQ ID NO: 2. Each combination of an N-terminal or C-terminal position as a fragment of, for example, at least 7 or at least 15 contiguous amino acid residues length could occupy the amino acid sequence of SEQ ID NO: 2 is included in the invention. Again, "of at least 7 contiguous amino acid residues in length" means 7 amino acid residues in length or any integer among 7 amino acids and the number of amino acid residues of the full length polypeptide of the present invention. Specifically, each and every one of the integer numbers between 7 and the number of amino acid residues of the full-length polypeptide in are included in the present invention. Polypeptides that carry epitopes, immunogenic and antigenic compounds of the invention are useful, for example, for making antibodies which bind specifically to the polypeptides of the invention and in immunoassays to detect the polypeptides of the present invention. The antibodies are useful, for example, in the affinity purification of the polypeptides of the present invention. Antibodies can also be used routinely in a variety of qualitative and quantitative immunoassays, specifically for the polypeptides of the present invention using methods known in the art. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press; 2nd Edition 1988). Epitope-bearing polypeptides of the present invention can be produced by any conventional means to make the polypeptides including synthetic and recombinant methods known in the art. For example, epitope-bearing peptides can be synthesized using known methods of chemical synthesis. For example, Houghten has described a simple method for the synthesis of large numbers of peptides, such as 10-20 mgs of 248 individuals and 13 different residue peptides representing the individual amino acid variants of a HAI polypeptide segment, all which were prepared and characterized (by ELISA-type binding studies) in less than four weeks (Houhgten, RA Proc. Nati, Acad. Sci. USA 82: 5131-5135 (1985)). This process of "Multiple Peptide Synthesis, Simultaneous (SMPS)" is further described in the North American patent No. 4,631,211 issued to Houghten et al. (1986). In this procedure, the individual resins for the solid phase synthesis of various peptides are contained in separate solvent permeable packages, which make possible the optimal use of the many, identical, repetitive steps involved in the solid phase methods. A completely manual procedure allows 500-1000 or more syntheses to be run simultaneously (Houghten et al. (1985) Proc. Nati. Acad. Sci. 82: 5131-5135 in 5134. The epitope-bearing polypeptides of the present invention are used for inducing the antibodies in accordance with methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and methods of visualizing bacteriophages. See e.g., Sutcliffe et al. supra, Wilson et al., supra, and Bittle, et al. (1985) J. Gen. Virol. 66: 2347-2354.If immunization is used in vivo, animals can be immunized with a free peptide; The anti-peptide antibody titre can be raised by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. cysteine residues can be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides can be coupled to the carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free peptides or coupled to the carrier, for example, by intraperitoneal and / or intradermal injection of emulsions containing approximately 100 μg of peptide or carrier protein and Freund's adjuvant. Various booster injections may be necessary, for example, at intervals of about two weeks, to provide a useful titre of anti-peptide antibody which can be detected, for example, by the ELISA assay using a free peptide adsorbed on a solid surface. The titre of anti-peptide antibodies in the serum of an immunized animal can be increased by the selection of anti-peptide antibodies, for example, by adsorption to the peptide on a solid support and elution of the antibodies selected in accordance with the methods well known in the art. As one of skill in the art will appreciate, and discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to the heterologous polypeptide sequences. For example, the polypeptides of the present invention can be fused to the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, any combination thereof including both complete domains and portions thereof) which results in the chimeric polypeptides. These fusion proteins facilitate purification and show an increased life duration in vi vo. This has been shown, for example, by the chimeric proteins consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, for example, EPA 0,394,827; Traunecker et al. (1988) Nature 331: 84-86. Fusion proteins having a dimeric structure linked to the disulfide, due to the IgG portion, may also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or single fragments thereof. See, for example, Fountoulakis et al. (1995) J. Biochem. 270: 3958-3964. The nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in the detection and purification of the expressed polypeptide.

Antibodies The present invention further relates to antibodies and T cell antigen (TCR) receptors which specifically bind the polypeptides of the present invention. The antibodies of the present invention include IgG (including IgG1, IgG2j, IgG3 and IgG4), IgA (including IgA1 and IgA2), IgD, IgE or IgM and IgY. As used herein, the term "antibody" (Ab) is proposed to include whole antibodies, including whole chain individual antibodies and antigen binding fragments thereof. Much more preferably, the antibodies are fragments of antigen, human binding antibodies of the present invention include, but are not limited to, Fab, Fab 'and F (ab') 2, Fd, single chain Fvs ( scFv), single chain antibodies, Fvs linked to the disulfide (sdFv) and fragments comprising either a V or VH domain. The antibodies can be of any animal origin including birds and mammals. Preferably, the antibodies are from human, murine, rabbit, goat, guinea pig, camel, horse or chicken. Fragments of antigen binding antibodies, which include the individual chain antibodies, may comprise the variable region (s) alone or in combination with all or part of the following: articulation region, CH1 domains , CH2 and CH3. Also included in the invention are any of the combinations of the variable region (s) and the articulation region, the CH1, CH2 and CH3 domains. The present invention also includes chimeric, humanized, and monoclonal and polyclonal antibodies of human which specifically bind the polypeptides of the present invention. The present invention also includes antibodies which are anti-idiotypic to the antibodies of the present invention. The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. The multispecific antibodies may be specific for the different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or a solid support material. See, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. and collaborators (1991) J. Immunol. 147: 60-69; U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S.A. and collaborators (1992) J. Immunol. 148: 1574-1553. The antibodies of the present invention can be described or specified in terms of the epitope (s) or portion (s) of a polypeptide of the present invention which are specifically recognized or bound by the antibody. The epitope (s) or polypeptide portion (s) can be specified as described herein, eg, by the N-terminal and C-terminal positions, by the size at the contiguous amino acid residues , or listed in the Tables and Figures. The antibodies which specifically bind any epitope or polypeptide of the present invention can also be excluded. Therefore, the present invention includes antibodies that specifically bind the polypeptides of the present invention, and allows the exclusion thereof. The antibodies of the present invention can also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog or homolog of the polypeptides of the present invention are included. Antibodies that do not bind polypeptides with 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 (calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. Also included in the present invention are antibodies which only bind the polypeptides encoded by the polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions. (as described herein). The antibodies of the present invention can also be described or specified in terms of their binding affinity. Preferred binding affinities include those with a dosing constants or Kd less than 5X10"6M, 10" 6M, 5X10"7M, 10" 7M, 5X10"8M, 10" 8M, 5X10"9, 10" 9M, 5X10" 10, 10"10M, 5X10_11M, 10_11M, 5X10" 12M, 10"12M, 5X10" 13M, 10"I3M, 5X10" 14M, 10"1M, 5X10" 15M and 10"15M. The antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target or target the polypeptides of the present invention that include the diagnostic and therapeutic methods both in vi tro and in vivo. For example, antibodies have use in immunoassays to qualitatively or quantitatively measure the levels of the polypeptides of the present invention in biological samples. See, for example, Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988) (incorporated herein by reference in the entirety). The antibodies of the present invention can be used either alone or in combination with other compositions. The antibodies can additionally be fused recombinantly to a heterologous polypeptide at the N or C terminus or can be chemically conjugated (including covalently and non-covalently conjugated) to the polypeptides or other compositions. For example, the antibodies of the present invention can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, for example, WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 0 396 387. The antibodies of the present invention can be prepared by any suitable method known in the art. For example, a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of recombinant technology and hybridomas. See, for example, Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling and collaborators, in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y, 1981) (references incorporated by reference in their entirety). The fragments of Fab and F (ab ') 2 can be produced by proteolytic cleavage, using enzymes such as papain (to produce the Fab fragments) or pesin (to produce the fragments of F (ab ') 2). Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art. For example, the antibodies of the present invention can be prepared using various methods of visualization of bacteriophages known in the art. In the methods of visual representation of bacteriophages, functional antibody domains are visually represented on the surface of a bacteriophage particle which carries the polynucleotide sequences that condition them. The bacteriophage with a desired binding property is selected from a repertoire or a library of combinatorial antibodies (eg, human or murine) by selection directly with the antigen, typically the antigen bound or captured to a solid or counting surface. The bacteriophages used in these methods are typically filamentous bacteriophages including fd and M13 with the domains of Fab, Fv or Fv antibodies stabilized with disulfide recombinantly fused to the protein of either gene III or gene VIII of bacteriophages. Examples of methods of visualizing bacteriophages that can be used to make the antibodies of the present invention include those described in Brinkman U. et al. (1995) J. Immunol. Methods 182: 41-50; Ames, R.S. and collaborators (1995) J. Immunol.

Methods 184: 177-186; Kettleborough, C.A. and collaborators (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 / GB91 / 01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patent Nos. 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, 5,780,225, 5,658,727 and 5,733,743 (references are incorporated by reference in their entirety). as described in the above references, after selection of the bacteriophage, the regions encoding the bacteriophage antibodies can be isolated and used to generate the complete antibodies, including human antibodies, or any other antigen binding fragment. desired, and can be expressed in any desired host that includes mammalian cells, insect cells, plant cells, yeast and bacteria. For example, techniques for recombinantly producing the Fab, Fab 'and F (ab') 2 fragments can also be employed using methods known in the art such as those described in WO 92/22324; Mullinax, R.L. and collaborators (1992) BioTechniques 12 (6): 864-869; and Sawai, H. and collaborators (1995) AJRI 34: 26-34; and Better, M. et al. (1988) Science 240: 1041-1043 (references are incorporated by reference in their entirety). Examples of techniques which can be used to produce single chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al. (1991) Methods in Enzymology 203: 46-88; Shu, L. and collaborators (1993) PNAS 90: 7995-7999; and Sherra, A. and collaborators (1988) Science 240: 1038-1040. For some uses, including the in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized or human antibodies. Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies, S.D. and collaborators (1989) J. Im unol. Methods 125: 191-202; and U.S. Patent No. 5,807,715. Antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0 239 400; WO 91/09967; U.S. Patent Nos. 5,530,101; 5,585,089), coating or re-isolating (EP 0 592 106; EP 0 519 596; Padlan EA, (1991) Molecular Immunology 28 (4/5): 489-498; Studnicka GM et al. (1994) Protein Engineering 7 (6): 805-814; Roguska MA et al. (1994) PNAS 91: 969 -973), and interspersed with chains (U.S. Patent No. 5,565,332). Human antibodies can be made by a variety of methods known in the art, including the methods of visualizing bacteriophages described above. See also, U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645 (references are incorporated by reference in their entirety). In addition, recombinantly fused or chemically conjugated antibodies (including conjugations either covalently or non-covalently) to a polypeptide of the present invention are included herein. The antibodies may be specific for antigens other than the polypeptides of the present invention. For example, the antibodies can be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for the receptors. of surface of cells, particular. Antibodies fused or conjugated to the polypeptides of the present invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, for example, Harbor et al. Supra and WO 93/21232; EP 0 439 095; Maramura, M. and collaborators (1994) Immunol. Lett, 39: 91-99; U.S. Patent No. 5,474,981; Gillies, S.O. and collaborators (1992) PNAS 89: 1428-1432; Fell, H.P. et al. (1991) J. Immunol. 146: 2446-2452 (references are incorporated by reference in their entirety). The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to the antibody domains other than the variable regions. For example, the polypeptides of the present invention can be fused or conjugated to a Fe region of the antibody, a portion thereof. The portion of the antibody fused to a polypeptide of the present invention may comprise the hinge region, the CH1 domain, the CH2 domain, and the CH3 domain or any combination of the total domains or portions thereof. The polypeptides of the present invention can be fused or conjugated to the above antibody portions to increase the in vivo lifetime of the polypeptides or for use in immunoassays using methods known in the art. The polypeptides can also be fused or conjugated to the above antibody portions to form the multimers. For example, the Fe portions fused to the polypeptides of the present invention can form dimers through the disulfide bond between the Fe portions. The higher multimeric forms can be made by fusing the polypeptides to the IgA and IgM portions. . Methods for fusing or conjugating the polypeptides of the present invention to portions of the antibody are known in the art. See, e.g., U.S. Patent 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. and coworkers (1991) PNAS 88: 10535-10539; Zheng, X.X. and collaborators (1995) J. Immunol. 154: 5590-5600; and Vil. H. et al. (1992) PNAS 89: 11337-11341 (references are incorporated by reference in their entirety). The invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor / ligand interactions with the pblipeptides of the invention either partially or completely. Both receptor-specific antibodies and ligand-specific antibodies are included. The specific antibodies of receptors are included, which do not prevent the binding of ligands but prevent the activation of the receptor. The activation of the receptor (i.e., signaling) can be determined by techniques described herein or otherwise known in the art. Also included are receptor-specific antibodies which both prevent ligand binding and receptor activation. Likewise, neutralization antibodies are included which bind the ligand and prevent the binding of the ligand to the receptor, as well as the antibodies that bind the ligand, due to this they prevent receptor activation, but they do not prevent ligand binding. to the receiver. In addition, antibodies that activate the receptor are included. These antibodies can act as agonists for all or less than all the biological activities affected by receptor activation mediated by the ligand. The antibodies may be specific as agonists or antagonists for the biological activities comprising the specific activities described herein. The above antibody agonists can be made using methods known in the art. See, for example, WO 96/40281; U.S. Patent No. 5,811,097; Deng, B. and collaborators (1998) blood 92 (6): 1981-1988; Chen, Z. et al. (1998) Cancer Res. 58 (16): 3668-3678; Harrop, J.A. and collaborators (1998) J. Immunol. 161 (4): 1786-1794; Zhu, Z. et al. (1998) Cancer Res. 58 (15): 3209-3214; Yoon, D.Y. and collaborators (1998) J. Immunol. 160 (7): 3170-3179; Prat, M. and collaborators (1998) J. Cell. Sci, 111 (Pt2) .237-247; Pitard, V. and collaborators (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. and collaborators (1995) Neuron 14 (4): 755-762; Muller, Y. A. et al. (1998) Structure 6 (9): 1153-1167; Bartunek, P. et al. (1996) Cytokine 8 (l): 14-20 (references are incorporated by reference in their entirety). The antibodies can also be used in an immunoassay to detect the presence of tumors in certain individuals. The enzyme immunoassay can be performed from the blood sample of an individual. High levels of VEGF-2 can be considered cancer diagnoses.

Truncated versions of VEGF-2 that are capable of interacting with non-cultured VEGF-2 can also be produced to form dimers that fail to activate endothelial cell growth, thereby inactivating endogenous VEGF-2. Or, the mutant forms of VEGF-2 form dimers by themselves and occupy the ligand binding domain of the appropriate tyrosine kinase receptors on the surface of target cells, but fail in the activation of cell growth. Alternatively, antagonists can be employed for the polypeptides of the present invention, which bind to the receptors to which a polypeptide of the present invention normally binds. The antagonists can be closely related proteins such that they recognize and bind to the receptor sites of the natural protein, however, these are inactive forms of the natural protein and due to this they prevent the action of VEGF-2 since the receptor sites they are busy. In these forms, the action of VEGF is prevented and the antagonists / inhibitors can be used therapeutically as an anti-tumor drug by occupying tumor receptor sites, which are recognized by VEGF-2 or by inactivation of VEGF- 2 same. Angiotonists / inhibitors can also be used to prevent inflammation due to the increased vascular permeability action of VEGF-2. Antagonists / inhibitors can also be used to treat the growth of solid tumors, diabetic retinopathy, psoriasis and rheumatoid arthritis. Antagonists / inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, eg, empirically, as described above. The present invention will be further described with reference to the following examples; however, it should be understood that the present invention is not limited to such examples. All parts or quantities are by weight, unless otherwise specified. In order to facilitate understanding of the following examples, certain methods and / or terms that occur frequently will be described. The "plasmids" are designated by a bottom box p preceded and / or followed by uppercase letters and / or numbers. The plasmids of initiation herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from the available plasmids according to published procedures. In addition, plasmids equivalent to those described are known in the art and will be apparent to a skilled, ordinary artisan. The "digestion" of DNA refers to the • catalytic division of DNA with a restriction enzyme that only acts on certain sequences in DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 mg of plasmid or a DNA fragment with approximately 2 units of enzyme is used in approximately 20 Fl of buffer. For the purposes of isolation of DNA fragments for the construction of plasmid, 5 to 50 mg of DNA are typically digested with 20 to 250 units of enzyme in a larger volume. Suitable buffers and substrate amounts for the particular restriction enzymes are specified by the manufacturer. Incubation times of approximately 1 hour at 37 ° C are commonly used, but may vary according to the supplier's instructions. After digestion, the reaction is directly subjected to electrophoresis in a polyacrylamide gel to isolate the desired fragment. The size separation of the divided fragments is carried out using 8 percent of the polyacrylamide gel described by Goeddel, D. et al., Nuclei c Acids Res. 8: 4051 (1980). The "oligonucleotides" refer to either an individual chain polydeoxynucleotide or two complementary polydeoxynucleotide chains, which can be chemically synthesized. Such synthetic oligonucleotides do not have 5 'phosphate and thus will not be ligated to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will be ligated to a fragment that has not been dephosphorylated. "Ligation" refers to the process for forming phosphodiester linkages between two double stranded nucleic acid fragments (Sambrook et al., Mol ecular Clinging: A Labora tory Manua l, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), p.146). Unless provided otherwise, ligation can be performed using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 mg of approximately equimolar amounts of the DNA fragments to be ligated. Unless stated otherwise, the transformation was performed as described by the method of Graham, F. and Van der Eb, A., Virol. Ogy 52: 456-457 (1973).

Examples Example 1 Expression Pattern of VEGF-2 in Human Tissues and Breast Cancer Cell Lines The Northern Blot analysis was carried out to examine the expression levels of the VEGF-2 in human tissues and breast cancer cell lines in human tissues. The samples of Cell RNA, complete were isolated with the system RNAzolMR B (Biotex Laboratories, Inc.). Approximately 10 mg of the complete RNA isolated from each breast tissue and cell line specified was separated on a 1% agarose gel and blotted on a nylon filter (Sambrook et al., Mol ecul a r oning: A Labora tory Manua l, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). The labeling reaction was done according to the Stratagene Prime-It kit with 50 ng of the DNA fragment. The labeled DNA was purified with a Select-G-50 column from 5 Prime-3 Prime, Inc. (Boulder, CO). The filter was then hybridized with a full length, labeled, radioactive VEGF-2 gene at 1,000,000 cpm / ml in 0.5 M NaP04 and 7% SDS overnight at 65 ° C. After washing twice at room temperature and twice at 60 ° C with 0.5 X SSC, 0.1% SDS, the filters were then exposed at -70 ° C overnight with an intensification screen. A 1.6 kd message was observed in 2 breast cancer cell lines. Figure 5, band # 4 represents a very tumorigenic cell line that is estrogen-independent for growth. Also, 10 mg of whole RNA was separated from adult human tissues in an agarose gel and treated with blotter paper on a nylon filter. The filter was then hybridized with the radiolabelled VEGF-2 test substance in 7% SDS, 0.5 M NaP04, pH 7.2; BSA 1% overnight at 65 ° C. After washing in 0.2X SSX at 65 ° C, the filter was exposed to the film for 24 days at -70 ° C with an intensification screen. See Figure 6.

E mplo 2 Expression of the Truncated Form of VEGF-2 (SEQ ID NO: 4) Through an In vitro Transcription and Translation The VEGF-2 cDNA was transcribed and translated in vi tro to determine the size of the translatable polypeptide, encoded by the truncated form of VEGF-2 and a partial VEGF-2 cDNA. The two VEFG-2 inserts in the pBluescript SK vector were amplified by PCR with three primer pairs, 1) forward and forward M13 primers; 2) M13-reverse primer and F4 primer of VEGF; and 3) M13-reverse primer and F5 primer of VEGF. The sequence of these primers are as follows. Inverse M13-2 primer: 5 '-ATGCTTCCGGCTCGTATG-3' (SEQ ID NO: 9). This sequence is located upstream of the 5 'end of the VEGF-2 cDNA insert in the pBluescript vector and is in an antisense orientation such as the cDNA. A T3 promoter sequence is located between this primer and the VEGF-2 cDNA. Primer M13-2 forward: 5 'GGGTTTTCCCAGTCACGAC-3' (SEQ ID NO: 10). This sequence is located downstream of the 3 'end of the VEGF-2 cDNA insert in the pBluescript vector and is in an antisense orientation as the cDNA insert.

VEGF primer F4: 5 'CCACATGGTTCAGGAAAGACA-3' (SEQ ID NO: 11). This sequence is located within the VEGF-2 cDNA in an antisense orientation of bp 1259-1239, which is approximately 169 bp away from the 3 'end of the stop codon and approximately 266 bp before the last nucleotide of the cDNA. The PCR reaction with all three pairs of primers gives the amplified products with the T3 promoter sequence in front of the cDNA insert. The first and third primer pairs give the PCR products encoding the VEGF-2 polypeptide shown in SEQ ID NO: 4. The second pair of primers gives the PCR product without the sequence encoding the 36 amino acids at the C terminus of the VEGF-2 polypeptide. Approximately 0.5 mg of the PCR product of the first pair of primers was used, 1 mg of the second pair of primers, 1 mg of the third pair of primers for transcription / translation in vi tro. The in vitro transcription / translation reaction was performed in 25 fl volume, using the TNTJ Coupled Reticulocyte Lysate Systems (Promega, CAT # L4950). Specifically, the reaction contains 12.5 Fl of rabbit reticulocyte lysate TNT, 2 Fl of TNT reaction buffer, 1 Fl of T3 polymerase, 1 Fl of mixture of 1 mM amino acid (less methionine), 4 Fl of 35 S-methionine ( >1000 Ci / mmol, 10 mCi / mL), 1 Fl of 40 U / μL; Inhibitor of RNasin ribonuclease, 0.5 or 1 mg of the PCR products. H20-free H20 was added to bring the volume to 25 Fl. The reaction was incubated at 30 ° C for 2 hours. Five microliters of the reaction product were analyzed on a gradient of 4-20% SDS-PAGE gel. After fixation in 25% isopropanol and 10% acetic acid, the gel was dried and exposed to an X-ray film overnight at 70 ° C. As shown in Figure 7, the PCR products containing the truncated VEGF-2 cDNA (ie, as depicted in SEQ ID NO: 3) and the cDNA without 266 bp in the 3 'untranslated region (3 '-UTR) produced the same length of translated products, whose molecular weights are estimated to be 38-40 dk (lanes 1 and 3). The cDNA without all 3'UTR and without the sequence encoding the C-terminal 36 amino acids was translated into a polypeptide with an estimated molecular weight of 36-38 kd (lane 2).

Example 3 cloning and Expression of VEGF-2 Using the Vaculovirus Expression System The DNA sequence encoding the VEGF-2 protein without 46 amino acids at the N-terminus, see ATCC No. 97149, was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene: The 5 'primer has the sequence TGT AAT ACG ACT CAC TAT AGG GAT CCC GCC ATG GAG GCC ACG GCT TAT GC (SEQ ID NO: 12) and contains a BamHl restriction enzyme site (in bold) and the 17 nucleotide sequence complementary to the 5 'sequence of VEGF-2 (nt 150-166). The 3 'primer has the sequence GATC TCT AGA TTA GCT CAT TTG TGG TCT (SEQ ID NO: 13) and contains the cleavage site for the restriction enzyme Xbal and 18 complementary nucleotides for the 3' sequence of VEGF-2, inclusive the stop codon and a sequence of 15 nt before the stop codon. The amplified sequences were isolated from a 1% agarose gel using commercially available equipment ("Geneclean", BIO 101, Inc., La Jolla, CA). The fragment was then digested with the endonuclease BamHI and Xbal and then purified again on a 1% agarose gel. This fragment was ligated to the baculovirus transfer vector pAcGP67A (Pharmingen) at the BamHl and Xbal sites. Through this ligand, the VEGF-2 cDNA was cloned into the structure with the signal sequence of the baculovirus gp67 gene and located at the 3 'end of the signal sequence in the vector. This is designated pAcGP67A-VEGF-2. To clone VEGF-2 with the gp67 gene signal sequence to the pRG1 vector for expression, VEGF-2 with the signal sequence and some upstream sequence was cut from the plasmid pAcGP67A-VEFG-2 at the endonuclease site Xho restriction site located downstream of the VEGF-2 cDNA and at the Xbal restriction endonuclease site by the restriction enzyme Xhol and Xbal. This fragment was separated from the rest of the vector on a 1% agarose gel and purified using a "Geneclean" kit. This was designated F2. The vector PRG1 (modification of vector pVL941) was used for the expression of the VEGF-2 protein using the baculovirus expression system (for review see: Summers, MD and Smith, GE, "A Man ual of Methods for Ba culovirus Vectors and Insect Cell Cul ture Procedures, "Texas Agricultural Experimental Station Bulletin No. 1555, (1987)). This expression vector contains the strong polyhedrin promoter of the nuclear polyhedrosis virus Au tographa california (AcMNPV) followed by the recognition sites for the restriction of the endonucleases BamHl, Smal, Xbal, BglII and Asp718. A site for the restriction of the Xhol endonuclease is located upstream of the BamHI site. The sequence between Xhol and BamHl is the same as that in the vector PAcGp67A (static in the tape). The polyadenylation site of simian virus (SV) 40 is used for efficient polyadenylation. For an easy selection of the recombinant virus, the E. coli beta-galactosidase gene is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked on both sides by the viral sequences for cell-mediated homologous recombination of the uncultured, contracted viral DNA. Many other baculovirus vectors could be used in place of pRG1 such as pAc373, pVL941 and pAcIMl (Luckow, VA and Summers, MD, Virology 1 70: 31-39 (1989).) The plasmid was digested with restriction enzymes Xbol and Xbal and then dephosphorylated using calf intestinal phosphatase by procedures known in the art.The DNA was then isolated from a 1% agarose gel using commercially available equipment ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA was designated V2.

The F2 fragment and the dephosphorylated plasmid V2 were ligated with T4 DNA ligase. The HB101 cells of E. coli were then transformed and the bacteria were identified as containing the plasmid (pBac gp67-VEGF-2) with the VEGF-2 gene using the enzymes BamHl and Xbal. The sequence of the cloned fragment was confirmed by ordering DNA sequences. 5 mg of the plasmid pBac gp67-VEGF-2 was cotransfected with 1.0 mg of a commercially available linearized baculovirus ("BaculoGoldJ baculovirus DNA", Pharmingen, San Diego, CA. ) using the lipofectin method (Felgner et al, Proc.Na.I Acad.Sci, USA 84: 7413-7417 (1987)). 1 mg of BaculoGoldJ virus DNA and 5 mg of plasmid pBac gp67-VEGF-2 were mixed in a sterile well of a microtiter plate containing 50 ml of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD) . Then 10 ml of Lipofectin plus 90 ml of Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then, the transfection mixture was added dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded on a 35 mm tissue culture plate with 1 ml of Grace's medium without serum. The plate was rocked from side to side to mix the recently added solution.

The plate was then incubated for 5 hours at 27 ° C. After 5 hours, the transfection solution was separated from the plate and 1 ml of Grace's insect medium supplemented with fetal calf serum 10% was added. The plate was again placed in an incubator and the culture was continued at 27 ° C for four days. After four days the supernatant was collected and plaque assayed in a similar manner as described by Summers and Smith, supra. As a modification, an agar gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used which allows easy isolation of the blue-stained plates. (A detailed description of a "plate assay" can also be found in the user guide for the culturing of insect cells and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10). Four days after the serial dilution, the virus was added to the cells, the blue stained plates were punctured with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 ml of Grace's medium. The agar was separated by brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect the Sf9 cells seeded in 35 mm dishes. Four days later, the supernatants from these culture dishes were harvested and then stored at 4 ° C. Sf9 cells were cultured in Grace's medium supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-gp67-VEGF-2 at a multiplicity of infection (MOI) of 1. Six hours later, the medium was separated and replaced with the SF900 II medium minus methionine and cysteine (Life Technologies Inc. ., Gaithersburg). 42 hours later, 5 mCi of 35S-methionine and 5 mCi of 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labeled proteins were visualized by SDS-PAGE and autoradiography. The medium protein and the cytoplasm of Sf9 cells were analyzed by SDS-PAGE under non-reducing and reducing conditions. See Figures 8A and 8B, respectively. The medium was dialyzed against 50 mM of MES, pH 5.8. The precipitates were obtained after dialysis and resuspended in 100 mM NaCitrate, pH 5.0. The resuspended pellet was analyzed again by SDS-PAGE and stained with Coomassie Brilliant Blue. See Figure 9.

The supernatant of the medium was also diluted 1:10 in 50 mM of 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. The protein was eluted with the progressive gradients in 200, 300 and 500 mM NaCl. VEGF-2 was obtained using elution at 500 mM. The eluate was analyzed by SDS-PAGE in the presence or absence of a reducing agent, b-mercaptoethanol and stained by Coomassie Brilliant Blue. See Figure 10.

EXAMPLE 4 Expression of VEGF-2 Recobinant in COS Cells Plasmid expression, VEGF-2-HA, was derived from a pcDNAI / Amp vector (Invitrogen) containing: 1) origin of duplication of SV40, 2) gene of resistance to ampicillin, 3) origin of duplication of E. col i, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site. A DNA fragment encoding the complete VEGF-2 precursor and an HA tag fused to the structure at its end 3 'was cloned into the polylinker region of the vector, therefore, the expression of recombinant proteins is directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (Wilson et al., Cell 37: 1 61 (1984)). Infusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope. The plasmid construction strategy is described as follows: The DNA sequence encoding VEGF-2, ATCC no. 97149, was constructed by PCR using two primers: the 5 'primer (CGC GGA TCC ATG ACT GTA CTC TAC CCA) (SEQ ID NO: 14) contains a BamH1 site followed by the 18 nucleotides of the sequence encoding the VEGF- 2 that start from the initiation codon; sequence 3 '(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) contains the complementary sequences for a Xbal site, an HA tag, an Xhol site and the latter nucleotides of the sequence encoding VEGF-2 (does not include the stop codon). Therefore, the PCR product contains a BamHI site, the coding sequence followed by an Xhol restriction endonuclease site and an HA tag fused to the structure, a stop coding, terminator, translation tag after tag of HA and an Xbal site. The DNA fragment amplified by the PCR and the vector, pcDNAI / Amp, were digested with the restriction enzyme BamHI and Xbal and ligated. The ligation mixture was transformed into the E chain. col i SURE (Stratagene Cloning Systems, La Jolla, CA 92037) the transformed culture was plated with ampicillin medium and resistant colonies were selected. Plasmid DNA was isolated from the transformants and examined by restriction analysis for the presence of the correct fragment. For expression of recombinant VEGF-2, COS cells were transfected with the expression vector by the DEAE-DEXTRAN method (J.

Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A labora tory Manua, Cold Spring Laboratory Press, (1989)). Expression of the VEGF-2-HA protein was detected by radiolabelling and the immunoprecipitation method (E. Harlow and D. Lane, An tibodi is: A Labora tory Manua, Cold Spring Harbor Laboratory Press, (1988)). The cells were labeled for 8 hours with 35S-cysteine two days after transfection. The culture media were then collected and the cells were used with detergent (RIPA buffer (150 mM NaCl, NP-40 1%, SDS 0.1%, NP-40 1%, DOC 0.5%, 50 mM Tris, pH 7.5) (Wilson et al., Cell 37: 767 (1984)) The media for both cell lysate and culture were precipitated with a monoclonal antibody, specific for HA.The precipitated proteins were analyzed on SDS-PAGE gels. %.

Example 5 The Effect of Protein of VEGF-2 Partially Purified on the Growth of Endothelial, Vascular Cells On day 1, human umbilical vein endothelial cells (HUVEC) were seeded at a density of 2-5x104 cells / 35 mm plate in the medium M199 containing 4% fetal bovine serum (FBS), 16 units / ml of heparin and 50 units / ml of endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units / ml heparin. The VEGF-2 protein of SEQ ID NO. 2 minus the initial 45 amino acid residues, (VEGF) and basic FGF (bFGF) were added, at the concentration shown. On days 4 and 6, the medium was replaced. On day 8, the number of cells was determined with a Coulter counter (See Figure 12).

Example 6 The Effect of Protein of Purified VEGF-2 on the Growth of Endothelial, Vascular Cells On day 1, endothelial cells of the human umbilical vein (HUVEC) were seeded at a density of 2-5 x 10 4 cells / 35 mm dish in an M199 medium containing 4% fetal bovine serum (FBS), 16 units / ml of heparin, 50 units / ml of endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium was replaced with M199 containing 10% FBS, 8 units / ml heparin. The purified VEGF-2 protein of SEQ ID NO: 2 minus the initial 45 amino acid residues was added to the medium at this point. On days 4 and 6, the medium was replaced with the new medium and the supplements. On day 8, the number of cells was determined with the Coulter Counter (See Figure 13).

Example 7 Expression by means of Gene Therapy Fibroblasts were obtained from a subject by skin biopsy. The resulting tissue was placed in a tissue culture medium and separated into small pieces. Small pieces of tissue were placed on a wet surface of a tissue culture flask, approximately ten pieces were placed in each flask. The flask was turned upside down, closed tightly and left at room temperature overnight. After 24 hours at room temperature, the flask was inverted and pieces of tissue fixed at the bottom of the flask were added and new media were added (eg Ham's F12 media, with 10% FBS, penicillin and streptomycin). This is then incubated at 37 ° C for about a week. At this time, new media are added and changed subsequently every several days. After two additional weeks in culture, a monolayer of fibroblasts emerges. The monolayer is treated with trypsin and enlarged in larger flasks. PMV-7 (Kirschmeier, PT et al., DNA 7: 219-225 (1988) flanked by long, terminal repeats of Moloney murine sarcoma virus is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated in agarose gel and purified using glass beads The cDNA encoding a polypeptide of the present invention is amplified using the PCR primers which correspond to the 5 'and 3' end sequences respectively The 5 'primer contains an EcoRI site and the 3' primer also includes a HindIII site Equal quantities of the linear, murine murine sarcoma Moloney virus strand and the amplified EcoRI and HindIII fragment are added together in the presence of T4 DNA ligase The resulting mixture is maintained under conditions suitable for the ligation of the two fragments.The ligation mixture is used to transform the HB101 bacteria, which go are plated on kanamycin plates containing agar for the purpose of confirming which vector had the gene of interest properly inserted. Amfotropic pA317 or GP + aml2 packaging cells are grown in a tissue culture at a confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce viral, infectious particles that contain the gene (the packaging cells are now referred to as producer cells). New media are added to the producer cells, transduced and subsequently, the media are harvested from a 10 cm plate of the confluent producer cells. The depleted media, which contain the viral, infectious particles, are filtered through a millipore filter to separate the separated producer cells and these media are then used to infect the fibroblast cells. The media is separated from a sub-confluent plate of fibroblasts and replaced rapidly with the media of the producer cells. These media are separated and replaced with new media. If the virus titer is high, then virtually all fibroblasts will be infected and a selection is not required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. The designed fibroblasts are then injected into the host, either alone or after being cultured for confluence in 3 cytodex microcarrier beads. Fibroblasts now produce the protein product.

EXAMPLE 8 Expression of VEGF-2 mRNA in Human, Fetus and Adult Tissues Experimental Design Northern blot analysis was carried out to examine the expression levels of VEGF-2 mRNA in human tissues of fetuses and adults. A cDNA test substance containing the complete nucleotide sequence of the VEGF-2 protein was labeled with 32P using the DNA labeling system rediprime ° (Amersham Life Science), according to the manufacturer's instructions. After labeling, the test substance was purified using a CHROMA SPIN-100 * column (Clontech Laboratories, Inc.), according to the manufacturer's protocol number PT-1200-1. The labeled, purified test substance was then used to examine the various human tissues for the VEGF-2 mRNA. A Multi-Tissue Northern blotting paper (MTN) containing various tissues of human (Fetal Kidney, Fetal Lung, Fetal Liver, Brain, Kidney, Lung, Liver, Spleen, Thymus, Bone Marrow, Testicles, Placenta and Skeletal Muscle) was obtained. ) of Clontech. The MTN blotter was examined with the labeled test substance using the ExpressHyb * hybridization solution (Clontech) according to the manufacturer's protocol number PT1190-1. After hybridization and washing, the blotting paper was exposed to a film at 70 ° C overnight with an intensification screen and developed according to normal procedures.

Results Expression of VEGF-2 mRNA is abundant in vascular smooth muscle and various highly vascularized tissues. VEGF-2 is expressed at significantly higher levels in tissues associated with hematopoietic or angiogenic activities, ie, fetal kidney tissue, fetal lung, bone marrow, placenta, spleen and lung tissue. The level of expression of VEGF-2 is low in the adult kidney, fetal liver, adult liver, testes; and it is almost undetectable in the fetal brain, and the adult brain (See Figure 14). In cells cultured first, expression of VEGF-2 mRNA is abundant in vascular smooth muscle cells and dermal fibroblast cells, but much lower in endothelial cells of the human umbilical vein (see Figure 15). This distribution pattern of mRNA is very similar to that of VEGF.

Example 9 Construction of amino terminal and carboxy terminal deletion mutations In order to identify and analyze the biologically active VEGF-2 polypeptides, a panel of VEGF-2 deletion mutations was constructed using the expression vector pHE4a. 1. Construction of VEGF-2 T103-L215 in pHE4 To allow the directed amplification by the Polymerase Chain Reaction and subcloning of VEGF-2 T103-L215 (amino acids 103 to 215 in Figure 1 or SEQ ID NO: 18) in the E. coli protein expression vector, pHE4, two primers were synthesized. oligonucleotides complementary to the desired region of VEGF-2 with the following sequence of bases. 'primer (Nde I / START and 18 nt of the cofferation sequence): 5' GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3 '(SEQ ID NO: 19) 3' primer (Asp718, STOP, and 15 nt of the coding sequence): '5' GCA GCA GGT ACC TCA CAG TTT AGA CAT GCA-3 '(SEQ ID NO: 20) The 5' primer described above (SEQ ID NO: 19), incorporates a restriction site Ndel and the 3 'Primer described above (SEQ ID NO: 20), incorporates an Asp718 restriction site. The 5 'primer (SEQ ID NO: 19) also contains an adjacent ATG sequence and in structure with the coding region of VEGF-2 to allow translation of the cloned fragment in E. coli, while the 3' primer (SEQ. ID NO: 20) contains a stop codon (preferentially used in E. coli) adjacent and in the structure with the coding region of VEGF-2 which ensures the completion of the correct translation in E. coli. The Polymerase Chain Reaction was performed using normal conditions well known to those skilled in the art and the nucleotide sequence for fully developed VEGF-2 (aa 24-419 in SEQ ID NO: 18), for example, constructed in Example 3 as a pattern. The resulting amplicon was digested by restriction with Ndel and Asp718 and subcloned into the expression vector pHE4a digested with Ndel / Asp718. 2. Construction of VEGF-2 T103-R227 in pHE4 To allow amplification directed by the Polymerase Chain Reaction and subcloning of VEGF-2 T103-227 (amino acids 103 to 227 in Figure 1 or SEQ ID NO: 8) in the E. coli protein expression vector, pHE4, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following sequence of bases. 'primer (Nde I / START and 18 nt of the coding sequence): 5' -GCA GCA CAT ATG ACÁ GAA GAG ACT ATA AAA-3 '(SEQ ID NO: 19) 3 'primer (Asp 718, DETENTION, and 15 nt of the coding sequence): 5' -GCA, GCA GGT ACC TCA ACG TCT AAT AAT GGA-3 '(SEQ ID NO: 21) In the case of the primers described above, an Ndel or Asp718 restriction site was incorporated into the 5 'primer and the 3' primer, respectively. The 5 'primer (SEQ ID NO: 19) also contains an adjacent ATG sequence and in the structure with the VEGF-2 coding region paxa pexmit the translation of the fragment cloned in E. coli, while the 3' primer ( SEQ ID NO: 21) contains a stop codon (preferentially used in E. coli) adjacent and in the structure with the coding region of VEGF-2 which ensures translation termination, correct in E. coli. The Polymerase Chain Reaction was performed using normal conditions well known to those skilled in the art and the nucleotide sequence for fully developed VEGF-2 (aa 24-419 in SEQ ID NO: 18), for example, constructed in Example 3, as a pattern. The resulting amplicon was digested by restriction with Ndel and Asp718 and subcloned into the protein expression vector pHE4a digested with Ndel / Asp718. 3. Construction of VEGF-2 T103-L215 in pA2GP In this illustrative example, the shuttle vector of plasmid pA2 GP is used to insert the cloned DNA encoding the suppressed VEGF-2 protein at the N-terminus and the C-terminus ( amino acids 103-215 in Figure 1 or SEQ ID NO: 18), in baculovirus to express the deleted VEGF-2 protein at the N-terminus and the C-terminus, using a baculovirus leader and normal methods as described in Summers et al., A Manual of Methods for Baculovirus Vector and Insect Cel l Cul ture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). This expression vector contains the strong polyhedrin promoter of the nuclear polyhedrosis virus Au tographa cali forni ca (AcMNPV) followed by the secretory signal peptide (leader) of the baculovirus gp67 protein and convenient restriction sites such as BamHl, Xba I and Asp718. The polyadenylation site of simian virus 40 ("SV40") is used for efficient polyadenylation. For easy selection of the recombinant virus, the plasmid contains the beta-galactosidase gene of E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked at both sites by the viral sequences for cell-mediated homologous recombination with viral ADJSI of the non-cultured type to generate viable viruses expressing the cloned polynucleotide. Many other baculovirus vectors could be used in place of the above vector, such as pAc373, pVL941 and pAcIMl, as one skilled in the art would readily appreciate, since the construct provides the appropriately localized signals for transcription, translation, secretion and the like , including a signal peptide and an AUG internal structure as required. Such vectors are described, for example, in Luckow et al., Virology 1 70: 31-39 (1989). The cDNA sequence encoding the VEGF-2 protein without 102 amino acids at the N-terminus and without 204 amino acids at the C-terminus in Figure 1, was amplified using PCR oligonucleotide primers corresponding to the sequences 5 'and 3' of the gene. The 5 'primer has the sequence 5' -GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA-3 '(SEQ ID NO: 22) containing the restriction enzyme site BamHl (in bold) followed by 1 nt separator to maintain the interior structure with the signal peptide supplied to the vector and 17 nt of the coding sequence bases of the VEGF-2 protein. The 3 'primer has the 5N-GCA sequence GCA TCT AGA TCA CAG TTT AGA CAT GCA-3' (SEQ ID NO: 23) containing the Xbal restriction site (in bold) followed by the detection codon and 17 complementary nucleotides for the 3 'coding sequence of VEGF-2. The amplified sequences were isolated from a 1% agarose gel using commercially available equipment ("Geneclean", BIO 101, Inc., La Jolla, CA).

The fxagment was then digested with the endonuclease BamHl and Xbal and then purified again on a 1% agarose gel. This fragment was ligated to the baculovirus transfer vector pA2 GP (Supplier) at the BamHl and Xbal sites. Through this ligation, the VEGF-2 cDNA representing the deleted VEGF-2 protein at the N-terminus and the C-terminus (amino acids 103-215 in Figure 1 or SEQ ID No. 18) was cloned in the structure with the signal sequence of the baculovirus GP gene and located at the 3 'end of the signal sequence in the vector. This is designated pA2GPVEGF-2.T103-L215. 4. Construction of VEGF-2 T103-R227 in pA2GP The cDNA sequence encoding the VEGF-2 protein without 102 amino acids at the N terminus and without 192 amino acids at the C terminus in Figure 1 (is decix, amino acids 103-227 of SEQ ID NO: 18) was amplified using PCR oligonucleotide primers corresponding to the 5 'and 3' sequences of the gene. The primer 5 '-GCA GCA GGA TCC CAC AGA AGA GAC TAT AAA ATT TGC TGC-3' has the sequence (SEQ ID NO: 24) containing the restriction enzyme site BamHl (in bold) followed by 1 nt separator for maintain the interior structure with the signal peptide supplied to the vector, and 26 nt of bases of the coding sequence of the VEGF-2 protein. The 3 'primer has the sequence 5M-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3' (SEQ ID NO: 25) containing the Xbal restriction site (in bold) followed by a stop codon and nucleotides complementary to the 3 'coding sequence of VEGF-2. The amplified sequences were isolated from a 1% agarose gel using commercially available equipment ("Geneclean", BIO 101, Inc., La Jolla, CA). The fxagment was then digested with the endonuclease BamH1 and Xbal and then purified again on a 1% agarose gel. This fragment was ligated to the baculovirus transfer vector pA2 GP (Supplier) at the BamHl and Xbal sites. Through this ligation, the VEGF-2 cDNA representing the deleted VEGF-2 protein at the N-terminus and the C-terminus (amino acids 103-227 in Figure 1 or SEQ ID NO: 18) was cloned into the structure with the signal sequence of the baculovirus GP gene and located at the 3 'end of the signal sequence in the vector. This construction is designated pA2GPVEGF-2. T103-R227.

. Construction of VEGF-2 in pCl The expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March 1985)) plus a fragment of the CMV enhancer (Boshart et al. Cell 42: 521-530 (1985)). Multiple cloning sites, for example, with the restriction enzyme cleavage sites BamHl, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors contain, in addition to the 3N intron, the polyadenylation and termination signal of the rat preproinsulin gene. The pCl vector is used for the expression of the VEGF-2 protein. Plasmid pCl is a derivative of plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under the control of the first SV40 promoter. The Chinese hamster ovary or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of DHFR genes in methotrexate-resistant cells (MXT) has been well documented (see, for example, Alt, FW, Kellems, RM, Bertino, JR, and Schimke, RT, 1978, J. Biol. Chem. 253: 1357-1370, Hamlin, JL and Ma, C. 1990, Biochem et Biophys, Acta, 1097: 101-143, Page, MJ and Sydenham, MA 1991, Biotechnology ogy 9: 64-68). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproduction of the target enzyme, DHFR, as a result of the amplification of the DHFR gene. If a second gene binds to the DHFR gene, it is usually co-amplified and over-expressed. It is a state of the art to develop cell lines that carry more than 1,000 copies of the genes. Subsequently, when methotrexate is removed, the cell lines contain the amplified gene integrated into the chromosome (s). Plasmid pCl contains for expressing the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al, Molecular and Cellular Biology, March 1985: 438-4470) plus a fragment isolated from the enhancer of the first human cytomegalovirus (CMV) immediate gene (Boshart et al., Cell 42: 521-530, 1985). Downstream of the promoter are the following individual restriction enzyme cleavage sites that allow the integration of the genes: BamHl, Pvull, and Nrul. Behind these cloning sites, the plasmid contains the stop codons of the translation in all three reading structures followed by the 3N intron and the polyadenylation site of the rat preproinsulin gene. Other efficient, high promoters can also be used for expression, for example promoters b-actin human, SV40 promoters or the first or subsequent repetitions long terminal from other retroviruses, eg, HIV and HTLVI. For polyadenylation of the mRNA, other signals can also be used, for example, of human growth hormone or globin genes. Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected on co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker at the start, for example, G418 plus methotrexate. Plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using intestinal calf phosphates by methods known in the art. The vector is then isolated from a 1% agarose gel. The DNA sequence encoding VEGF-2, ATCC Accession No. 97149, was constructed by PCR using two primers corresponding to the 5 'and 3' ends of the VEGF-2 gene: the 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)) contains a BamHI site filled in with Klenow and 40 nt of coding sequence of VEGF-2 start from the initiation codon; the 3 'primer (5' -GCA GGG TAC GGA TCC TAG ATT AGC TCA TTT GTG GTC TTT-3 '(SEQ ID NO: 27)) contains a BamHI site and 16 nt of the coding sequence of the _VEGF-2 not including the stop codon. The DNA fragment amplified with the PCR is isolated from a 1% agarose gel as described above and then digested with the BAmHI endonuclease and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with the T4 DNA ligase. The HB101 cells of E. coli are then transformed and bacteria containing the plasmid pCl are identified. The sequence and orientation of the inserted gene is confirmed by the ordering of DNA sequences. This construction is designated pClVEGF-2. 6. Construction of pC4SigVEGF-2 T103-L215 The plasmid pC4Sig is the plasmid pC4 (Accession No. 209646) which contains an IgG Fe portion of human as well as a sequence of protein signals. To allow amplification directed by the Polymerase Chain Reaction and subcloning of VEGF-2 T103-L215 (amino acids 103 to 215 in Figure 1 or SEQ ID NO: 18) in pC4Sig, two complementary oligonucleotide primers were synthesized for the desired region of VEGF-2 with The following sequence of bases: Primer 5 '(Bam Hl and 26 nt of the coding sequence): 5' -GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3 '(SEQ ID NO: 34) 3' primer (Xba I, STOP , and 15 nt of the coding sequence): 5 '-CGT CGT TCT AGA TCA CAG TTT AGA CAT GCA TCG GCA G-3' (SEQ ID NO: 35) The Polymerase Chain Reaction was performed using normal conditions well known to those skilled in the art and the nucleotide sequence for fully developed VEGF-2 (aa 24-419), for example, constructed in Example 3, as a standard. The resulting amplicon was digested by suppression with BamHI and Xbal and subcloned into the vector pC4Sig digested with BamHI / Xbal. 7. Construction of pC4SigVEGF-2 T103-R227 To allow targeted amplification by the Polymerase Chain Reaction and subcloning of VEGF-2 T103-L215 (amino acids 103 to 227 in Figure 1 or SEQ ID NO: 18) in pC4Sig, synthesized two oligonucleotide primers complementary to the desired region of VEGF-2 with the following sequence of bases: Primer 5 '(Bam Hl and 26 nt of the coding sequence): 5' -GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TTT GCT GC-3 '(SEQ ID NO: 34) 3' primer (Xba I, STOP , and 21 nt of the coding sequence): 5 '-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC-3' (SEQ ID NO: 25) The Polymerase Chain Reaction was performed using normal conditions well known for those skilled in the art and the nucleotide sequence for fully developed VEGF-2 (aa 24-419), for example, constructed in Example 3, as a standard. The resulting amplicon was digested by restriction with BamHI and Xbal and subcloned into the vector pC4Sig digested with BamHI / Xbal. 8. Construction of pC4VEGF-2 M1-M263 The expression vector pC4 contains the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV booster (Boshart et al., Cell 42: 521-530 (1985)). Multiple cloning sites, for example, with the restriction enzyme cleavage sites BamHl, Cbal and Asp718, facilitate the cloning of the gene of interest. The vector also contains the 3N intron, the polyadenylation signal and termination of the rat preproinsulin gene. In this illustrative example, the cloned DNA encoding the M1-M263 protein of VEGF-2 deleted at the C-terminus (amino acids 1-263 in Figure 2 or SEQ ID NO.18) is inserted into the plasmid vector pC4 for expressing the suppressed VEGF-2 protein at the C-terminus. To allow for the amplification directed by the Polymerase Chain Reaction and the subcloning of VEGF-2 M1-M263 into the expression vector, pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following sequence of bases. '5' Primer -GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3 '(SEQ ID NO: 28) Primer 3 '5' -GAC TGG TAC CTT-ATC ACA TAA AAT CTT CCT GAG CC-3 '(SEQ ID NO: 29) In the case of the 5 'primer described above, a BamHI restriction site was incorporated, whereas in the case of the 3' primer, an Asp718 restriction site was incorporated. The 5 'primer also contains 6 nt, 20 nt of the coding sequence of VEGF-2, and an adjacent ATG sequence and in structure with the coding region of VEGF-2 to allow translation of the cloned fragment in E. coli , while the 3 'primer contains 2 nt, 20 nt of the coding sequence of VEGF-2, a stop codon (used preferentially in E. coli) adjacent and in the structure with the coding region of VEGF-2 which ensures the completion of the correct translation in E. coli. The Polymerase Chain Reaction was performed using normal conditions well known to those skilled in the art and the nucleotide sequence for fully developed VEGF-2 (aa 24-419) constructed, for example, in Example 3 as a standard. The resulting amplicon was digested by restriction with BamHI and Asp718 and subcloned into the pC4 protein expression vector digested with BamHI / Asp718. This construction is designated pC4VEGF-2 Ml-M263. 9. Construction of pC4VEGF-2 M1-D311 In this illustrative example, the cloned DNA encoding the M1-D311 protein of VEGF-2 deleted at the C-terminus (amino acids 1-311 in Figure 1 or SEQ ID NO: 18) was inserted into a pC4 plasmid vector to express the suppressed VEGF-2 protein at the C-terminus. In order to allow the amplification directed by the Polymerase Chain Reaction and the subcloning of M1-D311 of VEGF-2 into the expression vector, pC4, two complementary oligonucleotide primers were synthesized for the desired region of VEGF-2 with the following sequence of bases: '5' Primer -GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3 '(SEQ ID NO: 30) Primer 3' 5- 'GAC TGG TAC CTT ATC AGT CTA GTT CTT TGT GGG G-3' (SED ID NO: 31) In the case of the 5 'primer described above, a BamHI restriction site was incorporated, whereas in the case of the 3' primer, an Asp718 restriction site was incorporated. The 5 'primer also contains 6 nt, 20 nt of a VEGF-2 coding sequence, and an adjacent ATG sequence and in structure with the coding region of VEGF-2 to allow translation of the fragment cloned in E. coli, whereas the 3 'primer contains 2 nt, 20 nt of the coding sequence of VEGF-2, and a stop codon (preferentially used in E. coli) adjacent and in the structure with the coding region of VEGF- 2 which ensures the completion of correct transduction in E. coli. The Polymerase Chain Reaction was performed using the normal conditions well known to those skilled in the art and the nucleotide sequence for fully developed VEGF-2 (aa 24-419) constructed, for example, in Example 3 as a standard. The resulting amplicon was digested by restriction with BamHI and Asp718 and subcloned into the pC4 protein expression vector digested with BamHI / Asp718.

. Construction of pC4VEGF-2 M1-Q367 In this illustrative example, the cloned DNA encoding the M1-D311 protein of VEGF-2_supressed at the C-terminus (amino acids 1-311 in SEQ ID NO: 18) is inserted into the vector of plasmid pC4 to express the VEGF-2 protein suppressed at the C-terminus. To allow amplification directed by the Polymerase Chain Reaction and subcloning of M1-D311 of VEGF-2 into the expression vector, pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following sequence of bases. '5' Primer -GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3 '(SEQ ID NO: 32) 3 '5' primer -GAC TGG TAC CTC ATT ACT GTG GAC TTT CTG TAC ATT C-3 '(SEQ ID NO: 33) In the case of the 5 'primer described above, a BamHI restriction site was incorporated, whereas in the case of the 3' primer, an Asp718 restriction site was incorporated. The 5 'primer also contains 6 nt, 20 nt of a VEGF-2 coding sequence, and an adjacent ATG sequence and in structure with the coding region of VEGF-2 to allow translation of the fragment cloned in E. coli, whereas the 3 'primer contains 2 nt, 20 nt of the coding sequence of VEGF-2, and a stop codon (preferentially used in E. coli) adjacent and in the structure with the coding region of VEGF-2 which ensures the completion of correct transduction in E. coli. The Polymerase Chain Reaction was performed using the normal conditions well known to those skilled in the art and the nucleotide sequence for fully developed VEGF-2 (aa 24-419) constructed, for example, in Example 3 as a standard. The resulting amplicon was digested with restriction with BamHI and Asp718 and subcloned into the pC4 protein expression vector digested with BamHI / Asp718. This construction is designated pC4VEGF-2 Ml-Q-367.

EXAMPLE 10 Transient Expression of VEGF-2 Protein in COS-7 Cells Experimental Design Expression of the VEGF-2-HA fusion protein of the construct made in Example 4, for example, was detected by the labeling ratio and the immunoprecipitation, using • the methods described in, for example, Harlow and colleagues (Antibodies: A Laboratory Manual, 2nd Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988)). To this end, two days after the transfection, the cells were labeled by incubation in media containing 35 S-cysteine for 8 hours. The cells and media were collected, and the cells were washed and then used with RIPA buffer containing detergent: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM of TRIS, pH 7.5, as described by Wilson and colleagues (supra). The proteins were precipitated from the cell lysate and from the culture media using a monoclonal antibody specific for HA. The precipitated proteins were then analyzed by SDS-PAGE and autoradiography.

Results As shown in Figure 16, cells transfected with pcDNA VEGF-2HA secreted a 56 kd and 30 kd pfotein. The 56 kg protein, but not the 30 kd protein, could also be detected in the cell lysate but was not detected in the controls. This suggests that the 30 kd protein is probably a result of the 56 kd protein split. Since the HA tag is at the C terminus of VEGF-2, the 30 kd protein must represent the C-terminal portion of the split protein, while the N-terminal portion of the split protein would not be detected - by immunoprecipitation. These data indicate that the VEGF-2 protein expressed in mammalian cells was secreted and processed.

Example 11: Stimulatory effect of VEGF-2 on the proliferation of endothelial, vascular cells Experimental Design The expression of VEGF-2 is abundant in highly vascularized tissues. Therefore, the role of VEGF-2 in the regulation of the proliferation of various types of endothelial cells was examined.

Endothelial Cell Proliferation Assay For the evaluation of the mitogenic activity of the growth factors, the assay of (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- ( 4-sulfophenyl) 2H-tetrazolium) of colorimetric MTS with the electron coupling reagent PMS (phenazine methosulfate) (CellTiter 96 AQ, Promega). The cells were seeded in a 96-well plate (5,000 cells / well) in 0.1 L of medium supplemented with serum and allowed to bind overnight. After starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF? 65 or VEGF-2 in 0.5% FBS) were added with or without Heparin (8 U / ml) to the wells for 48 hours. 20 mg of a mixture of MTS / PMS (1: 0.05) per well was added and allowed to incubate for 1 hour at 37 ° C before measuring the absorbance at 490 nm in an ELISA plate reader. The absorbance of the environment from the control wells (some media, without cells) was subtracted, and seven wells were performed in parallel for each condition. See, Leak et al. In Vi tro Cell. Dev. Biol. 30A: 512-518 (1994).

Results VEGF-2 slightly stimulated the proliferation of endothelial cells of the human umbilical vein (HUVEC) and microvascular, dermal endothelial cells (Figures 17 and 18). The stimulatory effect of VEGF-2 is more pronounced in the proliferation of endometrial and microvascular endothelial cells (Figure 19). Endothelial, endometrial (HEEC) cells demonstrated the greatest response to VEGF-2 (96% of the effect of VEGF on endothelial, microvascular cells). The response of endothelial, microvascular cells (HMEC) to VEGF-2 was 73% compared to VEGF. The response of HUVEC and BAEC (bovine aortic endothelial cells) to VEGF-2 was substantially lower in 10% and 7% respectively. The activity of the VEGF-2 protein has varied between the different purification runs with the stimulatory effect of certain batches in the proliferation of HUVEC which is significantly greater than that of the other batches.

Example 12 Inhibition of proliferation of vascular smooth muscle cells induced by PDGF The expression of VEGF-2 is high in vascular smooth muscle cells. Smooth muscle is a therapeutic target, important for vascular diseases, such as restenosis. To evaluate the potential effects of VEGF-2 on smooth muscle cells, the effect of VEGF-2 on the proliferation of human aortic smooth muscle cells (HAoSMC) was examined.

Experimental Design The proliferation of HAoSMC can be measured, for example, by the incorporation of BrdUrd. Briefly, quiescent, subconfluent cells cultured on 4-chamber slides were transfected with AT2-3LP labeled with CRP or FITC. Then, the cells are boosted with 10% calf serum and 6 mg / ml BrDUrd. After 24 hours, immunocytochemistry was performed using a BrdUrd staining kit (Zymed Laboratories). Briefly, the cells were incubated with the biotinylated mouse anti-BrdUrd antibody at 4 ° C for 2 hours after exposure to a denaturing solution and then with streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells were mounted for microscopic examination, and BrdUrd-positive cells were counted. The BrdUrd index is calculated as a percentage of BrdUrd-positive cells to the total number of cells. In addition, the simultaneous detection of BrdUrd staining (nucleus) and FITC uptake (cytoplasm) is carried out by individual cells by the concomitant use of bright field illumination and fluorescent illumination of dark field UV light. See, Hayashida et al., J. Biol. Chem. 6: 271 (36) -. 21985-21992 (1996).

Results VEGF-2 has an inhibitory effect on vascular smooth muscle cell proliferation induced by PDGF, but not by Bobino Fetal Serum (FBS) (Figure 20).

Example 13 stimulation of endothelial cell migration The migration of endothelial cells is an important step involved in angiogenesis. Experimental Design This example will be used to explore the possibility that VEGF-2 can stimulate the migration of endothelial, lymphatic cells. Currently, there are no published reports of a model of this kind. However, a model of endothelial, vascular cell migration for use with endothelial, lymphatic cells will be adapted essentially as follows: Endothelial cell migration assays were performed using a 48-well microchemistry 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. Immunologi cal Methods 1980; 33: 239-247). Free polyvinylpyrrolidone polycarbonate filters with a pore size of 8 um (Nucleopore Corp. Cambridge, MA) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. The test substances were diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution was placed in the lower chamber of the modified Boyden apparatus. The cultures of HUVEC or BMEC (2-6) first passage, subconfluents are washed and treated with trypsin for a minimum time required to achieve the cell section. After placing the filter between the lower and upper chamber, 2.5 x 105 cells suspended in 50 ul of M199 containing 1% FBS in the upper compartment are seeded. The apparatus is then incubated for 5 hours at 37 ° C in a chamber humidified with C02 5% to allow migration of the cells. After the incubation period, the filter separates and the upper side of the filter with the non-migrated cells is scraped with a rubber-tipped glass rod. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, IL). Migration is quantified by counting cells from three high-power, randomized fields (40x) in each well, and all groups were made in quadruplicate.

Results In an assay examining the migration of HUVEC using a 43-well microchemotaxis chamber, VEGF-2 was able to stimulate the migration of HUVEC (Figure 21).

Example 14 Stimulation of nitric oxide production by endothelial cells The nitric oxide released by the vascular endothelium is thought to mediate the relaxation of the vascular endothelium. It has been shown that VEGF-1 induces the production of nitric oxide by endothelial cells in response to VEGF-1. As a result, the activity of VEGF-2 can be tested by determining the production of nitric oxide by endothelial cells in response to VEGF-2.

Experimental Design Nitric oxide is measured in 96-well plates of microvascular endothelial cells, confluent after 24 hours of starvation and a subsequent exposure of 4 hours at various levels of the VEGF-1 and VEGF-2. Nitric oxide in the medium was determined by using Griess reagent to measure total nitrite after reduction of nitrate derived from nitric oxide by nitrate reductase. The effect of VEGF-2 on the release of nitric oxide was examined in HUVEC. In summary, the nitric oxide release of the cultured HUVEC monolayer was measured with a polarographic electrode, specific for nitric oxide connected to a nitric oxide meter (Iso-NO, World Precision Instruments Inc.) (1049). The calibration of the nitric oxide elements was carried out according to the following equation: 2 KN02 + 2 Kl + 2 H2S04 6 2 NO + I 2 + 2 H20 + 2 K2 S0 The normal calibration curve was obtained by adding graduated concentrations of KN02 (0, 5, 10, 25, 50, 100, 250 and 500 nmol / L) in the calibration solution containing Kl and H2S04. The specificity of the Iso-NO electrode to nitric oxide was previously determined by the nitric oxide measurement of authentic nitric oxide gas (1050). The culture medium was separated and the HUVECs were washed twice with saline buffered with Dulbecco's phosphate. The cells were then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates were maintained in a slide heater (Lab Line Instruments Inc.) to maintain the temperature at 37 ° C. The nitric oxide sensor probe was inserted vertically into the wells, keeping the tip of the electrode 2 mm below the surface of the solution, before the addition of the different conditions. Acetyl S-nitroso penicillamine (SNAP) was used as a positive control. The amount of nitric oxide released was expressed as picomoles by lxlO6 endothelial cells. All values reported were averages of four to six measurements in each group (number of cell culture wells). See, Leak and collaborators Biochem. and Biophys. Res. Comm. 21 7: 96-105 (1995).

Results VEGF-2 was able to stimulate the release of nitric oxide in HUVEC (Figure 22) to a higher level than VEGF. This suggests that VEGF-2 can modify vascular permeability and vessel dilation.

Example 15 Effect of VEGF-2 on cord formation in angiogenesis Another step in angiogenesis is cord formation, marked by the differentiation of endothelial cells. This bioassay measures the ability of endothelial, microvascular cells to form capillary-like structures (hollow structures) when grown in vi tro.

Experimental Design The CADMEC (endothelial, microvascular cells) are purchased from Cell Applications, Inc. as proligeration cells (passage 2) and grown in a CellMEC Growth Medium of Cell Applications and used in passage 5. For the assay of angiogenesis in vi tro, the wells of a 48-well cell culture plate are coated with a Cell Applications Binding Media Medium (200 ml / well) for 30 minutes at 37 ° C. The CADMEC are seeded in the coated wells at 7,500 cells / well and grown overnight in the Growth Medium. The Growth Medium is then replaced with 300 mg of a Cell Applications Cord-forming Medium containing the control buffer or HGS protein (0.1 to 100 ng / ml) and the cells are cultured for an additional 48 hours. The numbers and the lengths of the laces similar to the capillaries are quantified through the use of the video image analyzer Boeckeler VIA-170. All tests are done in triplicate. Commercial VEGF (R & D) (50 ng / ml) is used as a positive control, b-esteradiol (1 ng / ml) is used as a negative control. The appropriate buffer (without protein) is also used as a control.

Results It has been observed that VEGF-2 inhibits the formation of IFNa-like cords which also stimulate the proliferation of endothelial cells (Figure 23). This inhibitory effect may be a side effect of endothelial proliferation which is mutually exclusive with the cord formation process.

Example 16 Angiogenic effect on chorioallantoic membrane of chicks Chick chorioalonadal membrane (CAM) is a well-established system for examining angiogenesis. The formation of blood vessels in the CAM is easily visible and quantifiable. The capacity of VEGF-2 to stimulate angiogenesis in the CAM was examined.

Experimental Design Embryos Fertilized eggs of Leghorn chicks Whites (Gal lus ga ll us) and Japanese quail (Coturnix Coturnix were incubated at 37.8 ° C and 80% humidity.) The differentiated CAM of 16-day-old chicks and 13-day-old quail embryos were studied with The following methods.

CAM trial On day 4 of development, a window was made in the shell of the eggs of the chicks. The embryos were checked for normal development and the eggs were sealed with cellotape. These were further incubated until Day 13. The termanox coverslips (Nunc, Naperville, IL) were cut into discs approximately 5 mm in diameter. The sterile and salt-free growth factors were dissolved in distilled water and approximately 3.3 mg / 5 ml was pipetted into the disks. After drying with air, the inverted discs were applied in the CAM. After 3 days, the specimens were fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12 M sodium cacodylate buffer. These were photographed with a stereo microscope [Wild M8] and fixed for semi- and ultrathin sectioning as described above. The controls were performed with the carrier discs alone.

Results These data demonstrate that VEGF-2 can stimulate angiogenesis in the CAM assay nine times compared to the untreated control. However, this stimulation is only 45% of the level of VEGF stimulation (Figure 24).

EXAMPLE 17 Assay of angiogenesis using a Matrigel implant in a mouse Experimental Design In order to establish an in vivo model for angiogenesis to test the activities of the protein, mice and rats were implanted subcutaneously with methylcellulose disks containing either 20 mg BSA (negative control) and 1 mg of bFGF and 0.5 mg of VEGF-1 (positive control). This responded as it was thought, the discs of BSA contained little vascularization, whereas the discs of positive control showed signs of the formation of glasses. On day 9, a mouse showed a clear response to bFGF.

Results Both VEGF-2 proteins appeared to increase the cellularity of Matrigel by a factor of approximately 2 by visual appreciation.

Implants were made to 30 additional mice with discs containing BSA, bFGF, and varying amounts of VEGF-1, VEGF-2-B8 and VEGF-2-C4. Each mouse received two identical discs, preferably one control and one experimental disc. Samples of all recovered discs were immunostained with Von Willebrand factor to detect the presence of endothelial cells in the discs, and flK-1 and flt-4 to distinguish between endothelial, vascular and lymphatic cells. However, the histochemical, definitive analysis of neovascularization and lymphangiogenesis could not be determined.

Example 18 Recovery of Ischemia in a Lower Rabbit Member Model Experimental Design To study the in vivo effects of VEGF-2 on ischemia, a model of ischemia of the hind hind limb was created by surgically removing a femoral artery as previously described (Takeshita, S. et al., Am J. Pa thlol 247: 1649-1660 (1995)). The excision of the femoral artery results in the retrograde propagation of thrombi and occlusion of the external iliac artery. Consequently, blood flow to the ischemic member is dependent on collateral vessels originating from the internal iliac artery (Takeshita, S. et al. Am J. Pa thol 247: 1649-1660 (1995)). An interval of 10 days was left for the post-operative recovery of the rabbits and the development of collateral, endogenous vessels. In 10 days after the operation (day 0), after performing a baseline angiogram, the iliac, internal artery of the ischemic limb was transfected with 500 mg of pure VEGF-2 expression plasmid by arterial transfer technology of genes using a hydrogel-coated balloon catheter as described (Riessen, T. et al Hum Gene Ther 4: 749-758 (1993); Leclerc, G. et al., J. Clin. Invest., 90: 936- 944 (1993)). When VEGF-2 was used in the treatment, an individual bolus of 500 mg of the VEGF-2 protein or a control in the iliac artery, internal to the ischemic limb, was administered for a period of 1 minute through a catheter infusion. On day 30, various parameters were measured in these rabbits.

Results Both the VEGF-2 protein (Figure 25, upper panels) and the pure expression plasmid (Figure 25, middle panels) were able to restore the following parameters in the ischemic limb. The restoration of blood flow, the angiographic record seems to be slightly more by the administration of 500 mg of plasmid purchased with 500 mg of protein (Figure 25, background panels). The degree of restoration is comparable with that by VEGF in separate experiments (data not shown). A vessel dilator was not able to achieve the same effect, suggesting that the restoration of blood flow is not simply due to a vascular dilatation effect. to . PS ratio (Figure 25a) The blood pressure ratio of the systolic pressure of the ischemic limb to that of the normal limb. 2. Sagitic flow and Reserve of Flow (Figure 25b) FL at rest, blood flow during the undilated condition FL Max: blood flow during the fully dilated condition (also an indirect measurement of the amount of the blood vessel) the Flow Reserve reflects by the relation of the FL Max: FL in Rest. 3. Angiographic record (Figure 25c) This is measured by the angiogram of the collateral vessels. A record was determined by the percentage of circles in an overlying grid that with the intersection of the opacified arteries divided by the total number m the rabbit's thigh. '4. Capillary density (Figure 25d) The number of collateral capillaries determined in the light microscopic sections taken from the hind limbs.

As discussed, VEGF-2 is processed for an N-terminal and C-terminal fragment which is co-purified. The N-terminal fragment contains the functional, putative, intact domain and may be responsible for the biological activity.

Example 19 Effect of VEFG-2 on Vasodilation As described above, VEGF-2 can stimulate the release of nitric oxide, a mediator of vascular endothelial dilation. Since dilatation of the vascular endothelium is important in reducing blood pressure, the ability of VEGF-2 to affect blood pressure in spontaneously hypertensive rats (SHR) was examined. VEGF-2 caused a dose-dependent decrease in blood pressure, diastolic (Figures 26a and b). There was a regular decrease in blood pressure, diastolic with increasing doses of VEGF-2 which achieved statistical significance when a dosage of 300 mg / kg was administered. The changes observed in this dosage were not different from those observed with acetylcholine (0.5 mg / kg). An average, decreased blood pressure (MAP) was also observed (Figure 26c and d). VEGF-2 (300 mg / kg) and acetylcholine reduced the MAP of these SHR animals to normal levels.

Additionally, increasing dosages (0, 10, 30, 100, 300 and 900 mg / kg) of preparations B8, C5 and C4 of VEGF-2 were administered to spontaneously hypertensive rats (SHR) of 13-14 weeks of age. The data is expressed as the SEM +/- average. A statistical analysis was performed with an impaired t test and the statistical significance was defined as p < 0.05 against the response to the shock absorber alone. Studies with VEGF-2 (preparation C5) revealed that although blood pressure significantly decreased, the magnitude of the response was not as great as that observed with VEGF-2 (preparation B8) even when used in a dose of 900 mg / kg. Studies with VEGF-2 (preparation C4) revealed that this preparation of proteins expressed by CHO produced results similar to those observed with C5 (ie, statistically significant but much smaller than that observed with preparation B8) (see Figures 26A-D). As a control and since lots C4 and C5 of VEGF-2 produced minor but statistically significant changes in blood pressure, experiments were performed with another protein expressed by CHO, M-CIF. The administration of M-CIF at doses ranging from 10-900 mg / kg did not produce significant changes in blood pressure, diastolic. A statistically significant, lower reduction in mean arterial blood pressure was observed at doses of 100 to 900 mg / kg but no dose response was observed. These results suggest that the reductions in blood pressure observed with lots C4 and C5 of VEGF-2 were specific, ie, related to VEGF-2.

EXAMPLE 20 Rat Ischemic Skin Flap Model Experimental Design Evaluation parameters include skin t blood flow, skin temperature, and factor VIII immunohistochemistry or alkaline, endothelial phosphatase reaction. The expression of VEGF-2, during skin ischemia, is studied using in situ hybridization. The study in this model is divided into three parts as follows: a) Ischemic skin b) Ischemic skin wounds ~~ c) Normal wounds The experimental protocol includes: a) Produce a random skin flap, full thickness, individual pedicle , 3x4 cm (myocutaneous flap on the lower back of the animal). b) An excisional wound (4-6 mm in diameter) in the ischemic skin (skin flap). c) Topical treatment with VEGF-2 of the excisional wounds (day 0, 1, 2, 3, 4 after the wound) in the following different dose ranges: 1 mg to 100 mg. _ _ d) Collect wounded tissues on day 3, 5, 7, 10, 14 and 21 after wounding for histological, immunohistochemical and in situ studies.

Example 21 Model of Arterial Disease, Peripheral The angiogenic therapy that uses VEGF-2 has been developed as a new therapeutic strategy to obtain the restoration of blood flow around ischemia in case of arterial, peripheral diseases.

Experimental Design The experimental protocol includes: a) One side of the femoral artery is ligated to create an ischemic muscle of the hind limb, the other side of the hind limb serves as a control. b) the VEGF-2 protein, in a dose range of 20 mg-500 mg, is administered intravenously and / or intramuscularly 3 times (perhaps more) per week for 2-3 weeks. c) Ischemic muscle tissue is collected after ligation of the femoral artery in 1, 2 and 3 weeks for the analysis of VEGF-2 expression and histology. The biopsy is also performed on the other side of the normal contralateral, posterior limb muscle.

Example 22 Model of Myocardial Infarction, Ischemic VEGF-2 is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after occlusion of the coronary artery. The alteration of the expression of VEGF-2 is investigated in situ.

Experimental Design The experimental protocol includes: a) The heart is exposed through a left-sided toacostomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax closes. b) the VEGF-2 protein, in a dose range of 20 mg-500 mg, is administered intravenously and / or intramuscularly 3 times (perhaps more) per week for 2-4 weeks. c) Thirty days after surgery, the heart is separated and cut transversely for morphometric and in situ analysis.

Example 23 Rat Corneal Wound Healing Model This animal model shows the effect of VEGF-2 on neovascularization.

Experimental Design The experimental protocol includes: a) Mark an incision 1-1.5 mm long from the center of the cornea in the stromal layer. b) Insert a spatula under the edge of the incision in front of the outer cornea of the eye. c) Mark a receptacle (its base is 1-1.5 mm from the edge of the eye). d) Place a pellet, containing 50 mg - 500 mg of VEGF-2, inside the receptacle. e) The treatment of VEGF-2 can also be applied topically to corneal wounds in a dose range of 20 mg - 500 mg (daily treatment for five days).

Example 24 Healing Models of Diabetic Mouse Wounds and Glucocorticoid Reduced Wounds Experimental Design The experimental protocol includes: 1. Diabetic mouse model db + / db + To demonstrate that VEGF-2 accelerates the healing process, the genetically diabetic mouse wound healing model is used. The full-thickness wound healing model in the db + / db + mouse is a well-characterized, clinically relevant and reproducible model of impaired wound healing. The healing of diabetic wounds is dependent on the formation of the granulation tissue and the re-epithelialization preferably that the contraction (Gartner, MH et al, J. Surg. Res. 52: 389 (1992); Greenhalgh, DG et al., Am. J. Pathol. 136: 1235 (1990)). Diabetic animals have many of the characteristic qualities observed in Type II diabetes mellitus. The homozygous (db + / db +) mice are obese in comparison with their heterozygous, normal straw fluffy partners (db + / + m). Diabetic mice, mutations (db + / db +) have a recessive, autosomal, individual mutation on chromosome 4 (db +) (Coleman and collaborators Proc. Nati. Acad. Sci, USA 77: 283-293 (1982)). The animals show polyphagia, polydipsia and polyuria. Diabetic mice, mutants (db + / db +) have elevated blood glucose, increased or normal insulin levels, and immunity mediated by suppressed cells (Mandel et al., J. Immunol., 120: 1315 (1978); Debray-Sachs, M. et al. , Clin. Exp. Immunol. 51 (1): 1-7 (1983); Leiter et al., Am. J. of Pathol. 224: 46-55 (1985)). Peripheral neuropathy, myocardial complications and microvascular lesions, thickening of the basement membrane and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83 (2): 221- 232 (1984); Robertson et al., Diabe tes 29 (1): 60-67 (1980); Giacomelli et al., Lab Inves t. 40 (4): 4 60 -413 (1979); Coleman, D.L., Diabetes 31 (Suppl): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is insulin resistant analogous to human type II diabetes (Mandel et al, J. Immunol 120: 1315-1311 (1978)). The characteristics observed in these animals suggest that the cure in this model may be similar to the cure observed in human diabetes (Greenhalgh, et al., Am. J. of Pa thol. 136: 1235-124 6 (1990)).

Animals C57BL / KsJ (db + / db +), female, genetically diabetic mice and their heterozygous, non-diabetic straw fluffy partners (db + / + m) were used in this study (Jackson Laboratories). The animals were acquired at 6 weeks of age and were 8 weeks old at the start of the study. The animals were housed individually and received food and water ad libitum. All manipulations were performed using aseptic techniques. The experiments were conducted in accordance with the rules and guidance of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for Care and Use of Laboratory Animáis.

Surgical wounds The wound protocol was performed in accordance • with previously reported methods (Tsuboi, R. and Rifkin, D.B., J. Exp. Med. 272: 245-251 (1990)). In summary, on the day of the wound, the animals were anesthetized with an intraperitoneal injection of Avert na (0.01 mg / mL), 2, 2, 2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal was shaved and the skin was washed with 70% ethanol and iodine solution. The surgical area was dried with sterile gauze before the wound. Then a full thickness 8mm Jierida was created using a Keyes tissue punch. Immediately after the wound, the surrounding skin is gently stretched to eliminate the expansion of the wound. The wounds are left open for the duration of the experiment. The application of the treatment is given topically during 5 consecutive days that begin the day of the wound. Before the treatment, the wounds are gently cleaned with saline, sterile and gauze sponges. Wounds are examined visually and photographed at a fixed distance on the day of surgery and at intervals of two days later. The wound closure is determined by the daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a graded Majeson gauge. The wounds are considered cured if the granulation tissue is not more visible and the wound is covered by a continuous epithelium. VEGF-2 is administered using a range of different doses of VEGF-2, from 4 mg to 500 mg per wound per day for 8 days in the vehicle. Vehicle control groups received 50 mL of vehicle solution. Death was caused by euthanasia to the animals on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wounds and the surrounding skin were then collected for histology and immunohistochemistry. Tissue specimens were placed in buffered formalin, 10% neutral in the tissue cartridges between the biopsy sponges for further processing.

Experimental design Three groups of 10 animals each were evaluated (controls of 5 diabetics and 5 non-diabetics): 1) vehicle placebo control, 2) VEGF-2.

Area Measurement and Wound Closure The closure of the wound was analyzed by measuring the area on the vertical and horizontal axis and obtaining the square, total area of the wound. The contraction is then estimated by establishing the differences between the initial wound area (day 0) and that after treatment (day 8). The area of the wound on day 1 was 64 mm2, the corresponding size of the dermal punch. The calculations were made using the following formula: [Area opened on day 8] - [Area opened on day 1] / [Area opened on day 1] Histology The specimens are fixed in 10% buffered formalin and the blocks fixed in paraffin are sectioned perpendicular to the wound surface (5 mm) and cut using a micron or Reichert-Jung. Routine hematoxylin-eoxin (H & E) staining is performed on the cross sections of the bisected wounds. The histological examination of the wounds is used to assess whether the healing process and the morphological appearance of repaired skin is altered by treatment with KGF-2. This assessment included verification of the presence of accumulation of Y cells, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturation (Greenhalgh, D.G. et al., Am. J. Pa thol. 136: 1235 (1990)). A calibrated lens micrometer is used by a blinded observer.

Immunohistochemistry Re-epithelialization Tissue sections are stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using the ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. The growth of keratinocytes is determined by evaluating the degree of reepithelialization of the wound using a calibrated lens micrometer.

Cell Proliferation Marker The cell proliferation / cyclin nuclear antigen (PCNA) in skin specimens was demonstrated using an anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. The arrangement of these sections is based on the degree of proliferation on a scale of 0-8, the lower side of the scale that reflects a slight proliferation on the upper side that reflects intense proliferation.

Statistical Analysis The experimental data are analyzed using a deteriorated t-test. A p-value of < 0.05- is considered significant.

B. Rat Model Impaired by Steroids The inhibition of wound healing by steroids has been well documented in various in vi tro and in vivo systems (Wahl, SM Glucocorticoids and Wound healing, In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989), Wahl, SM et al., J. Immunol., 115: 476-481 (1975), Werb, Z. et al., J. Exp. Med. 247-1684-1694 (1978)). Glucocorticoids delay wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert, RH, et al., An. In tern. Med. 37: 701-705 (1952)), proliferation of fibroblasts, and synthesis of collagen (Beck, LS et al, Growth Fa ctors 5: 295-304 (1991); Haynes BF et al., J. Clin. Inves t 61: 103-1 91 (1978)) and the production of a transient reduction of the circulation of monocytes (Haynes, BF, et al., J. Clin. Inves t 61: 703-797 (1978); Wahl, SM, "Glucocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and Clinical Aspects. Academic Press, New York, pages 280-302 (1989)). Systemic administration of steroids to decrease wound healing is a well-established phenomenon in rats (Beck, LS et al, Growth Fa ctors 5: 295-304 (1991); Hynes, BF, et al. J. Clin. Invest. 61: 103-191 (1978); Wahl, SM, "Glixcocorticoids and wound healing", In: Antiinflammatory Steroid Action: Basic and clinical Aspects. Academic Press, New York, pages 280-302 (1989); Pierce, G.F. and collaborators, Proc. Na ti. Acad.

Sci. USA 85: 2229-2233 (1989)). To demonstrate that VEGF-2 can accelerate the healing process, the effects of topical, multiple applications of VEGF-2 on excisional, full-thickness skin wounds are evaluated in rats in which the healing has been diminished by the systemic administration of methylprednisolone.

Animals Sprague Dawley rats, male, young adults weighing 250-300 g (Charles River) are used Laboratories) in this example. The animals are acquired at 8 weeks of age and were 9 weeks old in the - Start of the study. The healing response of the rats is decreased by the systemic administration of methylprednisolone (17 mg / kg / rat intramuscularly) at the time of wounding. The animals are housed individually and received food and water ad l ibi tum.

All manipulations were performed using aseptic techniques. This study was conducted in accordance with the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animáis.

Surgical wounds The wound protocol is followed according to section A, above. On the day of injury, the animals are anesthetized with an intramuscular injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The dorsal region of the animal is shaved and the skin is washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze before the wound. A full thickness wound of 8 mm is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. The applications of the test materials are given topically once a day for 7 consecutive days starting on the day of the wound and subsequent to the administration of methylprednisolone. Before the treatment, the wounds are gently cleaned with saline, sterile and gauze sponges. The wounds are examined visually and photographed at a fixed distance on the day of the wound and at the end of the treatment. The closure of the wound is determined by the daily measurement on days 1-5 and on day 8 for the Figures. The wounds are measured horizontally and vertically using a graduated Jameson caliper. Wounds are considered cured if the granulation tissue is not more visible and the wound is covered by a continuous epithelium. VEGF-2 is administered using a different dose range of VEGF-2, from 4 mg to 500 mg per wound per day for 8 days in the vehicle. Vehicle control groups received 50 mL of vehicle solution. Death was caused by euthanasia to the animals on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wounds and the surrounding skin were then collected for histology. Tissue specimens are placed in buffered formalin, 10% neutral in the tissue cartridges between biopsy sponges for further processing.

Experimental design Four groups of 10 animals each were evaluated (5 with methylprednisolone and 5 with glucocorticoid): 1) Untreated group, 2) Vehicle placebo control 3) Groups treated with VEGF-2.

Area Measurement and Wound Closure The wound closure was analyzed by measuring the area on the vertical and horizontal axis and obtaining the total area of the wound. The closure is then estimated by establishing the differences between the area of the initial wound (day 0) and that after treatment (day 8). The area of the wound on day 1 was 64 mm2, the corresponding size of the dermal punch. The calculations are made using the following formula; [Area opened on day 8] - [Area opened on day 1] / [Area opened on day 1] Histology Specimens are fixed in 10% buffered formalin and blocks fixed in paraffin are sectioned perpendicular to the wound surface ( 5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin staining (H & E) was performed on the cross sections of the bisected wounds. The histological examination of the wounds allows the assessment of whether the healing process and the morphological appearance of the repaired skin were improved by treatment with VEGF-2. A calibrated lens micrometer was used by a blinded observer to determine the distance of the wound opening.

Statistical Analysis The experimental data are analyzed using a deteriorated t-test. A p-value of < 0.05 is considered significant.

Example 25 Fragments of Specific Peptides to Generate Monoclonal Antibodies of VEGF-2 Four specific peptides have been generated (designated SP-40, SP-41, SP-42 and SP-43). These will be used to generate the monoclonal antibodies to analyze the processing of VEGF-2. The polypeptides are shown below: 1. "SP-40": MTVLYPEYWKMY (amino acids 70-81 in SEQ ID NO: 18) 2. "SP-41": KSIDNEWRKTQSMPREV (amino acids 120-136 (observe mutation C-> S at position 131) in SEQ ID NO: 18) 3. "SP-42": MSKLDVYRQVHSIIRR (amino acids 212-227 in SEQ ID NO: 18) 4. "SP-42": MFSSDAGDDSTDGFHDI (amino acids 263-279 in SEQ ID NO: 18) Example 26 Animal Model of Linf adema The purpose of this experimental approach is to create an appropriate and consistent model of lymphedema to analyze the therapeutic effects of VEGF-2 on lymphagiogenesis and the re-establishment of the circulatory, lymphatic system in the hind limb of the rat. The effectiveness is measured by the swelling of the affected limb volume, the quantification of the amount of lymphatic vasculature, the total blood plasma protein and the histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of edema is continued for up to 3-4 weeks.

Experimental Procedure Before the start of surgery, a blood sample was taken for protein concentration analysis. Male rats weighing ~ 350 g are dosed with Pentobarbital. Subsequently, the right legs were shaved from the knee to the hip. The shaved area is cleaned with gauze moistened in 70% EtOH. Blood is drawn for the complete serum protein test. The circumferential and volumetric measurements were made before injecting dye on the legs after marking 2 measurement levels (0.5 cm above the heel, at the midpoint of the dorsal leg). The intradermal back of the legs, both right and left, is injected with 0.05 ml of Evan's Blue 1%. The circumference and volumetric measurements are then made after the dye injection in the legs.

Using the knee joint as a mark, an inguinal incision is made in the middle of the leg that allows the femoral vessels to be located circumferentially. Forceps or forceps and hemostats are used to dissect and separate skin flaps. After locating the femoral vessels, locate the lymphatic vessel that runs along the side and below the vessel (s). The lymphatic vessels, major in this area then coagulate electrically or are ligated with suture. Using a microscope, the muscles on the back of the leg (near semitendinosis and adductors) are abruptly dissected. Then the lymph node is located, popliteal. The 2 lymphatic vessels, proximal and the distal 2 and the distal blood supply of the popliteal node are then ligated by suturing. The lymphatic node, popliteus and any adipose tissue that accompanies it, is then separated by cutting the connective tissues. Care was taken to control any mild bleeding resulting from this procedure. After the lymphatic modes were occluded, the flaps of the skin are sealed using liquid skin (Vetbond) (AJ Buck). The edges of the separated skin were sealed to the underlying muscle tissue while leaving an opening of ~ 0.5 cm around the leg. The skin can also be attached by suture to the overlying muscle when necessary. To avoid infection, the animals are housed individually with mesh (without bed). The recovery of the animals was checked daily through the optimal edematous peak, which typically occurred on day 5-7. The edematous peak of the plate was then observed. To assess the intensity of lymphedema, the circumference and volumes of the 2 designated places on each leg were measured before the operation and daily for 7 days. The effect that plasma proteins have on lymphedema is also investigated and it is determined if the protein analysis is a useful test perimeter. The weights of both control and edematous members were evaluated in 2 places. The analysis was done in a blind way.

Circumference measurements: Under the short gaseous anesthetic to prevent movement of the limb, a cloth tape is used to measure the circumference of the limb. The measurements are made in the ankle bone and the dorsal leg by 2 different people, then those 2 readings were averaged. The readings were taken from both the control and edematous members.

Volumetric measurements: On the day of surgery, animals were anesthetized with Pentobarbital and subjected to a test before surgery. For daily volumetric measurements, the animals are under brief halothane anesthetic (rapid immobilization and rapid recovery), both legs are shaved and are also marked using a waterproof marker on the legs. The legs are first immersed in water, then they are placed in an instrument at each marked level, then measured by the Buxco edema (Chem / Victor) programming elements. The data is recorded by one person, while the other is submerging the member in the marked area.

Measurements of the blood plasma protein: Blood is drawn, centrifuged and the serum is separated before surgery and then at the conclusion for the comparison of the complete protein and Ca2 +.

Comparison of Member Weight: After extracting the blood, the animal is prepared for the collection of tissue. The limbs were amputated using a quillitin, then the experimental and control legs were cut in the ligature and weighed. A second weighing was done when the tibio-calcaneus joint was disarticulated and the foot was weighed. Histological preparations: The transverse muscle located behind the knee area (popliteal) was dissected and ordered in a metal mold, filled with freezeGel, immersed in cold methylbutane, placed in sample bags labeled in -80EC until sectioning . In sectioning, the muscle was observed under fluorescent microscopy by lymph nodes. Other immuno / histological methods are currently being evaluated.

Example 27 Treatment Method Using Gene Therapy for the Production of VEGF-2 Polypeptide - In Vivo Another aspect of the present invention is to use gene therapy methods in vivo to treat disorders, diseases and conditions. The gene therapy method refers to the introduction of pure nucleic acid (DNA, RNA, and antisense DNA or RNA) comprising VEGF-2 operably linked to a promoter in an animal to increase expression of VEGF-2. Such gene therapy and techniques and methods of delivery are known in the art, see, for example, WO 90/11092, WO 98/11779; U.S. Patent Nos. 5693622, 5705151, 5580859; Tabata H, et al. (1997) Cardiovasc. Res. 35 (3): 470-479, Chao, J et al. (1997) Pharma col. Res. 35 (6): 517-522, Wolff, J.A. (1997) Neuromuscul. Disord. 7 (5): 314-318, Schwartz, 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 constructs can be delivered by any method that delivers the injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). ). The polynucleotide constructs of VEGF-2 can also be delivered directly into the arteries. The VEGF-2 polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier. The term "pure" polynucleotide, DNA or RNA refers to sequences that are free of any delivery vehicle that acts to aid, promote, or facilitate entry into cells, including viral sequences, viral particles, liposome formulations , lipofectin or precipitation agents and the like. However the VEGF-2 polynucleotide can also be delivered in liposome formulations (such as those taught in Felgner PL et al. (1995) Ann. NY Acad. Sci. 772: 126-139 and Abdallah B. et al. (1995) Biol. Cell 85 (1): 1-7) which can be prepared by methods well known to those skilled in the art. Constructs of the VEGF-2 vector used in the gene therapy method are preferably constructs that will not integrate into the host genome nor contain the sequences that allow duplication. Different from other gene therapy techniques, a major advantage of the introduction of the pure nucleic acid sequences into the target cells is the transient nature of the synthesis of polynucleotides in the cells. Studies have shown that DNA sequences without duplication can be introduced into the cells to provide production of the desired polypeptide for periods of up to six months.

The construction of VEGF-2 can be delivered to the interstitial tissue space within an animal, including the muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, biliary bladder, stomach, intestine, testes, ovary, uterus, rectum, nervous system, eye, gland and connective tissue. The interstitial space of the tissues comprises the intercellular fluid, the matrix of mucopolysaccharides between the reticular fibers of tissues of the organs, the elastic fibers in the walls of the vessels or chambers, the collagen fibers of fibrous tissues, or that same matrix within of the muscle cells that sheathe the connective tissue or in the bone pool. It is similarly the space occupied by the plasma of the circulation and the lymphatic fluid of the lymphatic channels. These can be conveniently supplied by injection into the tissues comprising these cells. These are preferentially delivered and expressed in non-dividing, persistent cells which are differentiated, although delivery and expression can be achieved in undifferentiated or less completely differentiated cells, such as, for example, blood stem cells or fibroblasts of the skin. Preferably, these are delivered by direct injection into the artery. For the injection of pure polynucleotides, an effective dosage amount of DNA or RA? will be in the range of about 0.05 g / kg of body weight to about 50 mg / kg of body weight. Preferably, the dose will be from about 0.005 mg / kg to about 20 mg / kg and more preferably from about 0.05 mg / kg to about 5 mg / kg. Naturally, as the architect of ordinary experience will appreciate, this dose will vary according to the tissue site of the injection. The appropriate and effective dose of the nucleic acid sequence can be easily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of the tissues, or directly into the arteries. However, other parenteral routes can also be used, such as inhaling an aerosol formulation particularly for delivery to the lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, constructions of pure VEGF-2 can be delivered to the arteries during angioplasty by the catheter used in the procedure. The dose response effects of the construction of the VEGF-2 polypeptide, injected into arteries in vivo, are determined as follows. The standard DNA, suitable for the production of mRNA encoding VEGF-2, is prepared according to a normal recombinant DNA methodology. The standard DNA, which can be either circular or linear, is used either as pure DNA or made a complex with liposomes. The arteries of rabbits are then injected with various amounts of the standard DNA. The hindlimb ischemia in rabbits is surgically induced, as described in Example 18. Immediately after this, five different sites are injected directly into the adductor (2 sites), large medium (2 sites) and semimembranous (1 site) muscles. with the plasmid DNA encoding VEGF-2 using a 3 ml syringe and the acute 2 gauge was advanced through a small incision in the skin. Then the skin is closed using nylon 4.0. The ability to recover from ischemia of hind limbs is determined by measuring the number of capillaries in sections of the light microscope taken from treated hind limbs, compared with hind limbs, ischemic rabbits not treated, calf blood pressure measurement and the measurement of the intra-arterial Doppler pilot wire of the flow velocity (Takeshita et al., J. Clin.Inves t 93: 662-670 (1994)). The results of previous experimentation in rabbits can be used to extrapolate the appropriate doses and other treatment parameters in humans and other animals using the pure DNA of VEGF-2 polynucleotides.

Example 28 Treatment Method Using Gene Therapy - Ex vivo - A gene therapy method transplants fibroblasts, which are capable of expressing VEGF-2 polypeptides, in a patient. In general, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in the tissue culture medium and separated into small pieces. Small pieces of tissue were placed on a wet surface of a tissue culture flask, about ten pieces are placed in each flask. The flask is turned down, sealed and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the pieces of tissue remain fixed to the bottom of the flask and new media are added (eg, F12 media from Ham, with 10% FBS, penicillin and streptomycin). The flasks are then incubated at 37 degrees C for about a week. At this time, new media are added and subsequently changed every several days, after two additional weeks of culture, a monolayer of fibroblasts emerges. The monolayer is treated with trypsin and enlarged in larger flasks. PMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988)), flanked by the long terminal repeats of Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with intestinal phosphatase from calf. The linear vector is fractionated in the agarose gel and purified, using glass beads. The cDNA encoding VEGF-2 can be amplified using the PCR primers which correspond to the 5 'and 3' end sequences respectively as set forth in Example 1. Preferably, the 5 'primer contains a site EcoRI and the 3 'primer includes a HindIII site. Equal amounts of the linear, murine Moloney murine sarcoma virus linear strand and the amplified EcoRI and HindIII fragment are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for the ligation of the two fragments. The ligation mixture is then used to transform the HB101 bacteria, which are then plated onto kanamycin-containing agar for the purpose of confirming that the vector contains the properly inserted VEGF-2. The amphotropic pA317 or GP + aml2 packaging cells are grown in tissue culture at the confluent density in the Dulbecco Codified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The vector of the MSV containing the VEGF-2 gene is then added to the media and the packaging cells are transduced with the vector. The packaging cells now produce viral, infectious particles that contain the VEGF-2 gene (packaging cells are now referred to as producer cells). New media are added to the producer cells, transduced and subsequently, the media are harvested from a 10 cm plate of the confluent producer cells. The spent medium, which contained the viral, infectious particles, is filtered through the millipore filter to separate the producer cells, set aside and these media are then used to infect the fibroblast cells. The media is separated from a sub-confluent plate of fibroblasts and replaced rapidly with the media of the producer cells. These media are separated and replaced with new media. If the virus titer is high, then virtually all fibroblasts will be infected and selection is not required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine if the VEGF-2 protein is produced. The designed fibroblasts are then transplanted into the host, either alone or after being cultured for confluence in 3 cytodex microcarrier beads.

EXAMPLE 29 Treatment Method Using Homologous Recombination of Gene Therapy Another method of gene therapy according to the present invention involves operably associating the endogenous VEGF-2 sequence with a promoter by means of homologous recombination as described , for example, in U.S. Patent No. 5,641,670, filed June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Koller and collaborators, Proc. Na ti. Acad. Sci, USA 85: 8932-8935 (1989); and Zijlstra et al., Na ture 342: 435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than the desired one. Constructs of polynucleotides are made which contain a promoter and target sequences, which are homologous to the 5 'non-coding sequence of endogenous VEGF-2, flanking the promoter. The target sequence will be sufficiently close to the 5 'end of VEGF-2 so that the promoter will be operably linked to the endogenous sequence in the homologous recombination. The promoter and target sequences can be amplified using PCR. Preferably, the amplified promoter contains different restriction enzyme sites at the 5 'and 3' ends. Preferably, the 3 'end of the first target sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second target sequence contains the same restriction site as the 3' end. of the amplifier promoter. The amplified promoter and the amplified target sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and the digested target sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for the ligation of the two fragments. The size of the construct is fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation. In this Example, the polynucleotide constructs are administered as pure polynucleotides by means of electroporation. However, polynucleotide constructs can also be administered with agents that facilitate transfection, such as liposomes, viral sequences, viral particles, precipitation agents, and the like. Such delivery methods are known in the art. Once the cells are transfected, homologous recombination will take place, which results in the promoter being operably linked to the endogenous VEGF-2 sequence. This results in the expression of VEGF-2 in the cell. Expression can be detected by immunological staining, or any other method known in the art. Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM + fetal calf serum 10%. The growth fibroblasts spontaneously or from the first stationary phase are treated with trypsin and rinsed from the plastic surface with a nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HP0, 6 mM dextrose). The cells are centrifuged, the supernatant is aspirated, and the cells are resuspended in the electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. The final cell suspension contains approximately 3X106 cells / ml. Electroporation should be performed immediately after resuspension. The plasmid DNA is prepared according to the normal techniques. To build a plasmid to target or target the VEGF-2 site, the pUCld plasmid (MBI Fermentáis, Amherst, NY) is digested with HindIII. The CMV promoter is amplified by PCR with a Xbal site at the 5 'end and a BamHI site at the 3' end. The two non-coding sequences of VEGF-2 were amplified by means of PCR: a non-coding sequence of VEGF-2 (fragment 1 of VEGF-2) is amplified with a HindIII site at the 5 'end and an Xba site on the 3 'end; the other non-coding sequence of VEGF-2 (fragment 2 of VEGF-2) is amplified with a BamH1 site at the 5 'end and a HindIII site at the 3' end. The CMV promoter and VEGF-2 fragments are digested with the appropriate enzymes (CMV promoter-Xbal and BamHI, fragment 1 of VEGF-2-Xbal, fragment 2 of VEGF-BamHI) and ligated together. The resulting ligation product is digested with HindIII and ligated with the pUC18 plasmid digested with HindIII. The plasmid DNA was added to a sterile specimen with a 0.4 cm electrode opening (Bio-Rad). The final DNA concentration is generally at least 120 μg / ml. Then, 0.5 ml of the cell suspension (containing approximately 1.5X106 cells) is added to the specimen, and the cell suspension and the DNA solutions are mixed gently. The electroporation is carried out with a Gene-Pulser device (Bio-Rad). The capacitance and voltage are adjusted to 960 μF and 250-300 V, respectively. When the voltage increases, the survival of the cells decreases, but the percentage of surviving cells that stably incorporate the DNA introduced into their genome increases dramatically. Given these parameters, an impulse time of approximately 14-20 mSec should be observed. _ The electroporated cells are kept at room temperature for about 5 minutes and the contents of the test piece are then gently separated with a sterile transfer pipette. The cells are added directly to 10 ml of the nutrient media, preheated (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 ° C. The next day, the media is aspirated and replaced with 10 ml of new media and incubated for 16-24 hours. The fibroblasts are then injected into the host, either alone or after being cultured for confluence in cytodex microcarrier beads. Fibroblasts now produce the protein product.

Example 30 Transgenic Animals of VEGF-2 The VEGF-2 polypeptides can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, for example, baboons, monkeys, and chimpanzees can be use to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express the polypeptides of the invention in humans, as part of a gene therapy protocol. Any technique known in the art can be used to introduce the transgene (ie, the polynucleotides of the invention) into the animals to produce the founder lines of the transgenic animals. Such techniques include, but are not limited to, pronuclear 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., U.S. Patent No. 4,873,191 (1989)); gene transfer mediated by retroviruses in the germ lines (Van der Puttern et al., Proc. Nati, Acad. Sci, USA 82: 6148-6152 (1985)), blasts or embryos; the target gene in embryonic stem cells (Thompson et al., Cell 56: 313-321 (1989)); the electroporation of cells or embryos (Lo, 1983, Mol Cell, Bio 3: 1803-1814 (1983)); the introduction of the polynucleotides of the invention using a gene gun (see, for example, Ulmer et al., Science 259: 1745 (1993); the introduction of nucleic acid constructs into stem, pluripotent, embryonic cells and the transfer of the stem cells again in the blastocyst, and the gene transfer mediated by sperm (Lavitran et al., Cell 57: 717-723 (1989), etc. For a review of such techniques, see Gordon, "Transgenic Animáis", Intl. Rev. Cytol. 115-: 171-229 (1989), which is incorporated herein by reference in its entirety.) Any technique known in the art can be used to produce the transgenic clones containing the polynucleotides of the invention. , for example, the nuclear transfer in nucleus-free oocytes of embryonic, fetal or cultured adult cell nuclei, induced for the resting state (Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997)). The present invention provides transgenic animals that carry the transgene in all their cells, as well as animals that carry the transgene in some, but not all of their cells, that is, mosaic or chimeric animals. The transgene can be integrated as an individual transgene or as multiple copies such as in concatamers, eg empl, tandems head to head or tandems head to tail. The transgene can also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Nati. Acad. Sci. USA 89: 6232-3636 (1992) ). The regulatory sequences required for such specific activation to the cell type will depend on the particular cell type of interest, and will be apparent to those skilled in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, targeting or gene targets are preferred.

In summary, when a technique of this kind is to be used, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integration, by means of homologous recombination with chromosomal sequences, in and the disruption of function of the nucleotide sequence of the endogenous gene. The transgene can also be selectively introduced into a particular cell type, to inactivate the endogenous gene in this type of cell in this way, by following, for example, the teaching of Gu et al. (Gu et al., Science 265: 103-106 (1994)). The regulatory sequences, required for this specific inactivation of the cell type, will depend on the particular cell type of interest, and will be apparent to those skilled in the art. Once the transgenic animals have been generated, the expression of the recombinant gene can be assayed using standard techniques. The initial selection can be performed by analysis with Southern blotting paper or PCR techniques to analyze the tissues of animals to verify that transgene integration has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals can also be assessed using techniques including, but not limited to, Northern blot analysis of the tissue samples obtained from the animal, the hybridization analysis in if you and the PCR-transcriptase (rt-PCT). Samples of the tissue expressing the transgenic gene can also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product. Once the founding animals are produced, they can be bred, bred within the same breed, bred by mixtures of breeds or bred as hybrids to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: breeding by breed mixtures of the founding animals with more than one integration site in order to establish separate lines; breeding within the same race of separate lines in order to produce transgenic compounds that express the transgene at the highest levels due to the effects of the expression of additives of each transgene; the crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both increase expression and eliminate the need to select animals by DNA analysis; the crossing of homozygous lines, separated to produce heterozygous or homozygous lines; and breeding to place the transgene in a different environment that is appropriate for an experimental model of interest. The transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in the development of the biological function of the VEGF-2 polypeptides, the study of conditions and / or associated disorders. with the expression of aberrant VEGF-2, and the selection of effective compounds in the improvement of these conditions and / or disorders.

Example 31 Animals Sensitized with VEGF-2 Endogenous VEGF-2 gene expression can also be reduced by inactivating or "sensitizing" the VEGF-2 gene and / or its promoter using homologous target recombination. (For example, see Smithies et al, Nature 317: 230-234 (1985), Thomas &Capecchi, Cell 51: 503-512 (1987), Thompson et al., Cell 5: 313-321 (1989); one of which is incorporated by reference in the present in its totatlidad). For example, a mutant non-functional polynucleotide of the invention (or a non-fully related DNA sequence) flanked by the homologous DNA for the endogenous polynucleotide sequence (either the coding regions or the regulatory regions of the gene) can be used, with or without a selectable marker and / or a selectable, negative marker, for transfecting the cells expressing the polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate sensitizations in cells that contain, but do not express, the gene of interest. The insertion of the DNA construct, by means of objective homologous recombination, results in the inactivation of the target gene. Such approaches are particularly suitable in research and agricultural fields where modifications to embryonic stem cells can be used to generate offspring of animals with a target gene, inactive (eg, see Thomas &Capecchi 1987 and Thompson 1989, supra). However, this approach can usually be adapted for use in humans provided that the recombinant DNA constructs are administered directly or target or target the required site in vivo using the appropriate viral vectors that will be apparent to those skilled in the art. The technique. In the further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered to not express the polypeptides of the invention (e.g., sensitizations) are administered to a patient in vivo Such cells can be obtained from the patient (i.e., animal, including human) or an MHC-compatible donor and can include, but are not limited to, fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, and so on. The cells are genetically engineered in vi tro using recombinant DNA techniques to introduce the coding sequence of the polypeptides of the invention into the cells, or alternatively, to disrupt the cloning sequence and / or regulatory, endogenous sequence associated with the polypeptides. of the invention, for example, by transduction (using viral vectors, and preferably the vectors that integrate the transgene into the genome of cells) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, pure DNA, electroporation, liposomes, and so on. The coding sequence of the polypeptides of the invention can be placed under the control of a strong or inducible constitutive promoter or a promoter / enhancer to achieve the expression, and preferably secretion, of the VEGF-2 polypeptides. The engineered cells that express, and preferentially secrete, the polypeptides of the invention can be introduced into the patient systemically, for example, in the circulation, or intraperitoneally. Alternatively, cells can be incorporated into a matrix and implanted in the body, for example, genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al., US Patent No. 5,399,349; and Mulligan & Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by reference herein in its entirety). When the cells to be administered are cells not derived from themselves or not compatible with HMC, they can be administered using well-known techniques that prevent the development of a host immune response against the introduced cells. For example, the cells can be introduced in an encapsulated form which, while allowing an exchange of components with the extracellular, immediate environment, does not allow the introduced cells to be recognized by the host's immune system. The sensitized animals of the invention have uses which include, but are not limited to, animal model systems useful in the development of the biological function of the VEGF-2 polypeptides, the study of conditions and / or disorders associated with the expression of aberrant BEGF-2, and in the selection of effective compounds in the improvement of such conditions and / or disorders. Numerous modifications and variations of the present invention are possible in view of the above teachings, and therefore, within the scope of the appended claims, the invention may be practiced differently as particularly described. The complete description of all publications (including patents, patent publications, journal articles, laboratory manuals, books or other documents) cited herein are hereby incorporated by reference).

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: Human Genome Sciences, Inc. (ii) TITLE OF THE INVENTION: ENDOTHELIAL GROWTH FACTOR, VASCULAR 2 (iii) NUMBER OF SEQUENCES: 35 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESS: HUMAN GENOME SCIENCES, INC. (B) STREET: 9410 KEY WEST AVENUE (C) CITY: ROCKVILLE (D) STATE: MARYLAND (E) COUNTRY: USA (F) ZIP: 20850 (v) READABLE COMPUTER FORM: (A) MEDIA TYPE: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) LOGICAL EQUIPMENT: Patentln Relay # 1.0, Version # 1.30 [vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: TO BE ASSIGNED (B) DATE OF PRESENTATION: WITH THE SAME (C) CLASSIFICATION: [vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 09 / 042,105 (B) DATE OF SUBMISSION: MARCH 13, 1997 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 09 / 107,997 (B) DATE OF SUBMISSION: JUNE 30, 1997 (C) CLASSIFICATION: (viii) INFORMATION OF THE APPORTER / AGENT (A) NAME: MICHELE M. WALES (B) REGISTRATION NUMBER: 43,975 (C) REFERENCE NUMBER / REGISTRATION: PF112PCT3 ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (301) 309-8504 (B) TELEFAX: (301) -309-8439 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1674 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: sig peptide (B) LOCATION: 12..80 (ix) CHARACTERISTICS: (A) NAME / KEY: mat_peptide (B) LOCATION: 81..1268 (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 12..1268 (xi) DESCRIPTION OF THE SEQUENCE: SEC I D NO: 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 Ala Leu Pro Gly Pro Arg Glu Ala Ala Ala Ala Ala -10 -5 1 5 GCC GCC TTC GAG TCC GCC CTC GAC CTC TCG GAC GCG GAG CCC GAC GCG 146 Wing Wing Phe Glu Ser Gly Leu Asp Leu Ser Asp Wing Glu Pro Asp Wing 10 15 20 GGC GAG GCC GCT ACCT GAT TAT GCA AGC AAA GAT CTG GAG GAG CAG TTA CGG 194 Gly Glu Wing Thr Wing Tyr Wing 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 lie Lys Phe 75 80 85 GCT GCA CAT TAT AAT ACÁ GAG ATC TTG AAA AGT ATT GAT AAT GAG 386 Ala Ala Ala His Tyr Asn Thr Giu He Leu Lys Ser He 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 He 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 HAV 578 Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu He Thr 155 160 165 GTG CCT CTC TCT CAA GGC CCC AAA CCA GTA ACÁ ATC AGT TTT GCC AAT 626 Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr He 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 ACÁ CTA CCA CAG TGT CAG 722 His Ser He He Arg Arg Ser Leu Pro Wing 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 Wing Wing Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His He 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 Wing Gln Glu Asp Phe Met Phe Ser Ser Asp Wing 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 He 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 Wing Gly Leu Arg Pro 265 270 275 GCC AGC TGT GGA CCC CAC AAA GAA CTA GAC AGA AAC TCA TGC CAG TGT 962 Wing Ser Cys Gly Pro His Lys Glu Leu Asp Ar g Asn Ser Cys G n 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 Wing 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 ACÁ GAA AGT 1106 Asn Gln Pro Leu Asn Pro Gly Lys Cys Wing Cys Glu Cys Thr Glu Ser 330 335 340 CCA CAG AAA TGC TTG TTA AAA GGA AAG AAG TTC CAC CAA 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 TGC ACG AAC CGC CAG AAG GCT TGT GAG CCA 1202 Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Wing 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 CTAACftAAGA 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 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 419 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE, protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Met His Ser Leu Gly Phe Phe Ser Val Wing Cys Ser Leu Leu Wing Ala -23 -20 -15 -10 Wing Leu Leu Pro Gly Pro Arg Glu Wing Pro Wing Wing Wing 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 Wing Asn Leu Asn Ser Arg Thr Glu Glu Thr He Lys Phe Ala Ala Ala 75 80 85 His Tyr Asn Thr Glu He Leu Lys Ser He Asp Asn Glu Trp Arg Lys 90 95 100 105 Thr Gln Cys Met Pro Arg Glu Val Cys lie 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 He Thr Val Pro Leu 155 160 165 Ser Gln Gly Pro Lys Pro Val Thr He Ser Phe Wing Asn His Thr Ser 170 175 180 185 Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser He 190 195 200 He Arg Arg Ser Leu Pro Wing Thr Leu Pro Gln Cys Gln Wing Wing Asn 205 210 215 Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His He Cys Arg Cys 220 225 230 Leu Wing Gln Glu Asp Phe Met Phe Ser Ser Asp Wing Gly Asp Asp Ser 235 240 245 Thr Asp Gly Phe His Asp He 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 Wing 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 Wing 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 (2) INFORMATION FOR SEQ ID NO: 3 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1526 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: sig_peptide (B) LOCATION: 71..142 (ix) CHARACTERISTICS: (A) NAME / KEY: mat_peptide (B) LOCATION: 143..1120 (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 71..1120 (xi) DESCRI PC OF THE S ECUENC IA: SEQ ID NO: 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 AC GAA GAG ACT ATA AAA TTT GCT GCA GCA CAT TAT 205 Leu Asn Ser Arg Thr Glu Glu Thr He Lys Phe Ala Ala Ala His Tyr 10"15 20 AAT ACÁ GAG ATC TTG AAA AGT ATT GAT AAT GAG TGG AGA AAG ACT CAA 253 Asn Thr Glu He Leu Lys Ser He 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 lie 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 Wing 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 Cyß Met Asn Thr Ser Thr 70 75 80 85 AGC TAC CTC AGC AAG ACG TTA TTT GAA ATT ACÁ GTG CCT TCTC CAA 445 Ser Tyr Leu Ser Lys Thr Leu Phe Glu He 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 He Ser Phe Wing 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 He lie 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 Wing 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 He 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 Wing 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 He 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 Wing Gly Leu Arg Pro Wing 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 AC 877 Leu Phe Pro Ser Gln Cys Gly Wing 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 ACÁ 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 CAAC ACÁ 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 Wing Cys Glu Pro Gly Phe Ser Tyr Ser 295 300 305 GAA GAA GTG TGT TGT TGT TGTC 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 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 350 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 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 Wing Asn Leu Asn Ser -5 1 5 Axg Thr Glu Glu Thx He Lys Phe Wing Wing His His Tyr Asn Thr Glu. 10 15 20 He Leu Lys Ser He Asp Asn Glu Trp Arg Lys Thr Gln Cys Met Pro 25 30 35 40 Arg- Glu Val Cys lie 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 He Thr Val Pro Leu Ser Gln Gly Pro Lys 90 95 100 Pro Val Thr lie 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 He He Arg Arg Ser Leu 125 130 135 Pro Wing Thr Leu Pro Gln Cys Gln Wing Wing Asn Lys Thr Cys Pro Thr 140 145 150 Asn Tyr Met Trp Asn Asn His He Cys Arg Cys Leu Wing Gln Glu Asp 155 160 165 Phe Met Phe Ser Ser Asp Wing Gly Asp Asp Ser Thr Asp Gly Phe His 170 175? 80 Asp He Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys 185 190 195 200 Val Cys Arg Wing Gly Leu Arg Pro Wing 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 Wing Asn Arg Glu Phe Asp Glu Asn Thr Cys Gln Cys 235 240 245 Val Cys Lys Arg T r 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 Wing Cys Glu Pro Gly Phe Ser Tyr Ser Glu Glu Val 300"305 310 Qys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met Ser 315 320 325 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 196 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5 Met Arg Thr Leu Wing Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Wing 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu He Pro Arg Glu Val He Glu Arg 20 25 30 Leu Ala Arg Ser Gln He His Ser Arg Asp Leu Gln Arg Leu Leu 35 40 45 Glu Xle Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg 50 55 60 Wing His Gly Val His Wing Thr Lys His Val Pro Glu Lys Arg Pro Leu 65 70 75 80 Pro He Arg Arg Lys Arg Ser He Glu Glu Ala Val Pro Ala Val Cys 85 90 95 Lys Thr Arg Thr Val He Tyr Glu He Pro Arg Ser Gln Val Asp Pro 100 105 110 Thr Ser Wing Asn Phe Leu He 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 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 241 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu A 1 5? o 15 Leu Val Ser Ala Glu Gly Asp Pro He Pro Glu Glu Leu Tyr Glu M 20 25 30 Leu Ser Asp His Ser He Arg Ser Phe Asp Asp Leu Gln Arg Leu L 35 40 45 Hxs Gly Asp Pro Gly Glu Glu Asp Gly Wing Glu Leu Asp Leu Asn M 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly A 65 70 75 8 Arg Ser Leu Gly Ser Leu Thr He Wing Glu Pro Wing Met He Wing G 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu He Ser Arg Arg Leu He A 100 105 lio Arg Thr Asn Wing Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val G 115 120 125 Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro T 130 135 140 Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys He Glu He Val A 145 150 155? Lys Lys Pro He Phe Lys Lys Wing Thr Val Thr Leu Glu Asp His Leu 165 170 '175 Wing Cys Lys Cys Glu Thr Val Wing Wing Wing Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly Gly Ser Gln Glu Gln Arg Wing Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr He 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 To (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 223 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 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 Wing Lys Trp Ser Gln Wing Wing Pro Met Wing 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 He Glu Thr Leu Val Asp He Phe Gln Glu 50 55 60 Tyr Pro Asp Glu He Glu Tyr He 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 Be Asn He Thr Met Gln He Met Arg He Lys Pro His 100 105 110 Gln Gly Gln His He Gly Glu Met Ser Phe Leu Gln His Asn Lys Cye 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 Wing Arg Cys Cys Leu Met Pro Trp 165 170 175 Ser Leu Pro Gly Pro Hxs Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 '185 190 Hxs 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 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: not relevant (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE :. SEC ID NO: Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn 1 5 10 2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA 'xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 ATGCTTCCGG CTCGTATG (2) INFORMATION FOR SEQ ID NO: 10 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10 GGGTTTTCCC AGTCACGAC 18 (2) INFORMATION FOR SEQ ID NO: 11: CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ; xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11 CCACATGGTT CAGGAAAGAC A 21 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12 TGTAATACGA CTCACTATAG GGATCCCGCC ATGGAGGCCA CGGCTTATGC 50 (2) INFORMATION FOR SEQ ID NO: 13 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13 GATCTCTAGA TTAGCTCATT TGTGGTCT 28 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14 CGCGGATCCA TGACTGTACT CTACCCA 27 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15 CGCTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAC TCGAGGCTCA TTTGTGGTCT 60 2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3974 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: both (D) TOPOLOGY: both (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 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 GTTTTTTCCAT AGGCTCCGCC 480 CCCCTGACGA GCATCACRAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 5 0 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 1550 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 2ISO 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 24SO 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 35 0 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 CCCAA'TTGTG AGCGGATAAC AATTTCACAC ATTAAAGAGG 3960 AGAAATTACA TATG 3974 2) INFORMATION FOR SEQ ID NO: 17: i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 112 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: both (D) TOPOLOGY: both (i i) T I PO OF MOLECULE: DNA (xi) DESCRI PC OF THE SECTION IA: SEC I D NO: 17 AAGCTTAAAA AACTGCAAAA AATAGTTTGA CTTGTGAGCG GATAACAATT AAGATGTACC 60 CAATTGTGAG CGGATAACAA TTTCACACAT TAAAGAGGAG AAATTACATA TG 112 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 419 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: Met His Ser Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Wing Ala 1 5 10 15 Wing Leu Leu Pro Gly Pro Arg Glu Wing Pro Wing Wing Wing Wing Wing Phe 20 25 30 Glu Ser Gly Leu Asp Leu Ser Asp Wing 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, Wing Asn Leu Asn Ser Arg Thr Glu Glu Thr He, Lys Phe Wing Wing 100 '105 110 is Tyr Asn Thr Glu He Leu Lys Ser He Asp Asn Glu Trp Arg Lys 115 120 125 Thr Gln Cys Met Pro Arg Glu Val Cys He Asp Val Gly Lys Glu Phe 130 135 140 Gly Val Wing 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 He Thr Val Pro Leu 180 185 190 Ser Gln Gly Pro Lys Pro Val Thr He Ser Phe Wing Asn His Thr Ser 195 200 205 Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser He 210 215 220 He Arg Arg Ser Leu Pro Wing Thr Leu Pro Gln Cys Gln Wing Ala Asn 225 230 235 240 Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His He Cys Arg Cys 245 250 255 Leu Wing Gln Glu Asp Phe Met Phe Ser As As Wing Gly Asp Asp Ser 260 265 270 Thr Asp Gly Phe His Asp He Cys Gly Pro Asn Lys Glu Leu Asp Glu 275 280 285 Glu Thr Cys Gln Cys Val Cys Arg Wing 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 Wing 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 Wing 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 Wing 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 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (i i) T I PO OF MOLECULE: cDNA (xi) DESCRI PC OF THE SEQUENCE: SEC I D NO: 19 GCAGCACATA TGACAGAAGA GACTATAAAA 30 (2) INFORMATION FOR SEQ ID NO: 20 i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20 GCAGCAGGTA CCTCACAGTT TAGACATGCA 30 (2) INFORMATION FOR SEQ ID NO: 21 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21 GCAGCAGGTA CCTCAACGTC TAATAATGGA 30 (2) INFORMATION FOR SEQ ID NO: 22 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: GCAGCAGGAT CCCACAGAAG AGACTATAAA 30 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ; xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23 GCAGCATCTA GATCACAGTT TAGACATGCA 30 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24 GCAGCAGGAT CCCACAGAAG AGACTATAAA ATTTGCTGC 39 INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25 GCAGCATCTA GATCAACGTC TAATAATGGA ATGAAC 36 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26 GATCGATCCA TCATGCACTC GCTGGGCTTC TTCTCTGTGG CGTGTTCTCT GCTCG 55 .2) INFORMATION FOR SECTION ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27 GCAGGGTACG GATCCTAGAT TAGCTCATTT GTGGTCTTT 39 [2) INFORMATION FOR SEQ ID NO: 28 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28 GACTGGATCC GCCACCATGC ACTCGCTGGG CTTCTTCTC 39 (2) INFORMATION FOR SEQ ID NO: 29 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29 GACTGGTACC TTATCACATA AAATCTTCCT GAGCC 35 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30 GACTGGATCC GCCACCATGC ACTCGCTGGG CTTCTTCTC 39 2) INFORMATION FOR SEQ ID NO: 31 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31 GACTGGTACC TTATCAGTCT AGTTCTTTGT GGGG 34 (2) INFORMATION FOR SEQ ID NO: 32 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32 GACTGGATCC GCCACCATGC ACTCGCTGGG CTTCTTCTC 39 (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear Ü i) T I PO OF MOLECULE: cDNA (xi) DESCRI PC OF SECUENC IA: SEC I D NO: 33 GACTGGTACC TCATTACTGT GGACTTTCTG TACATTC 37 (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34: GCAGCAGGAT CCACAGAAGA GACTATAAAA TTTGCTGC 38 (2) INFORMATION FOR SEQ ID NO: 35 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35 CGTCGTTCTA GATCACAGTT TAGACATGCA TCGGCAG 37

Claims (26)

1. An isolated polynucleotide, characterized in that it comprises at least 95% amino acid sequence identical to a polynucleotide which encodes a polypeptide selected from the group consisting of: (a) an N-terminal deletion fragment described by general formula m-396 of SEQ ID NO: 2; (b) a C-terminal deletion fragment described by general formula -23-n of SEQ ID NO: 2; (c) a N-terminal and C-terminal deletion fragment described by the general formula m-n of SEQ ID NO: 2; and (d) a C-terminal deletion fragment described by general formula + 9-n of SE ID NO: 2.

2. The isolated polynucleotide according to claim 1, characterized in that the polypeptide comprises the 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

3. A composition, characterized in that it comprises the isolated polynucleotide of claim 1.

4. The isolated polynucleotide according to claim 1, characterized in that the polynucleotide encodes a biologically active fragment of VEGF-2.

5. The isolated polynucleotide according to claim 1, characterized in that the polynucleotide encodes a polypeptide which binds an antibody to VEGF-2.

6. The polynucleotide according to claim 1, characterized in that it further comprises a polynucleotide which encodes a heterologous polypeptide.

7. A vector, characterized in that it comprises the polynucleotide according to claim 1.

8. The vector according to claim 7, characterized in that the polynucleotide is operatively associated with a regulatory, heterologous sequence.

9. A host cell, characterized in that it comprises the vector according to claim 7 or the polynucleotide of claim 1.

10. A method for producing a VEGF-2 polypeptide, characterized in that it comprises: (a) culturing the host cell according to claim 9 under conditions suitable for producing the polypeptide; and (b) recovering the polypeptide from the cell culture.

11. The polypeptide produced by the method according to claim 10.

12. An isolated polypeptide, characterized in that it comprises the at least 95% polypeptide identical to an amino acid sequence selected from the group consisting of: (a) an N-terminal deletion fragment described by the general formula m-396 of SEQ ID NO: 2; (b) a C-terminal deletion fragment described by general formula -23-n of SEQ ID NO: 2; (c) an N-terminal and C-terminal deletion fragment described by the general formula m-n of SEQ ID NO: 2; and (d) a C-terminal deletion fragment described by the general formula + 9-n of SEQ ID NO: 2.

13. The isolated polypeptide according to claim 12, characterized in that the polypeptide comprises the amino acid residues S-205 to S-396 of SEQ ID NO: 2 and / or the amino acid residues F-9 to R-203 of the SEC ID NO: 2

14. A composition, characterized in that it comprises the isolated polypeptide according to claim 1.

15. A composition, characterized in that it comprises a first fragment of polypeptides comprising the amino acid residues S-205 to S-396 of SEQ ID NO: 2 and a second fragment of polypeptides comprising amino acid residues F-9 to R-203 of SEC ID NO: 2.

16. The isolated polypeptide according to claim 12, characterized in that the polypeptide is a biologically active fragment.

17. The polypeptide isolated according to claim 12, characterized in that the polypeptide is antigenic.

18. The polypeptide isolated according to claim 12, characterized in that it also comprises a heterologous polypeptide.

19. An antibody to the polypeptide according to claim 12. __

20. A compound, characterized in that it activates the polypeptide according to claim 12.

21. A compound, characterized in that it inhibits the polypeptide according to claim 12.

22. A method for preventing, treating or ameliorating a medical condition, characterized in that it comprises administering to a mammalian subject a therapeutically effective amount of the polypeptide of claims 12-13, the composition of claims 3, 14-15, or of the polynucleotide of the claim 1-2.

23. A method for diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to the expression or activity of a secreted protein, characterized in that it comprises: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1; (b) diagnose a pathological condition or a susceptibility to a pathological condition based on the presence or absence of the mutation.

24. A method for diagnosing a pathological condition or a susceptibility to a pathological condition in a subject related to the expression or activity of a secreted protein, characterized in that it comprises: (a) determining the presence or amount of expression of the polypeptide of claim 12 in a biological sample; (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

25. A method for identifying the binding partner for the polypeptide of claim 10, characterized in that it comprises: (a) contacting the polypeptide of claim 12 with a binding partner; and (b) determining whether the binding partner carries out a polypeptide activity.

26. A method for identifying an activity in a biological assay, wherein the method comprises: (a) expressing the polypeptide of claim 12 in a cell; (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identify the protein in the supernatant that has the activity.