MXPA01007982A - Vascular endothelial growth factor-2 - Google Patents

Abstract

The present invention is directed to VEGF-2 polynucleotides and polypeptides and methods of using such polynucleotides and polypeptides. In particular, provided are methods of treating retinal disorders with VEGF-2 polynucleotides and polypeptides.

Description

FACTOR 2 OF VASCULAR ENDOTHELIAL GROWTH Background of the Invention The. present invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such 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 human vascular endothelial growth factor (VEGF2). The invention also relates to the inhibition of the action of such polypeptides. Additionally, the present invention relates to antibodies directed to the polypeptides of the present invention. The present invention also relates to the administration of polynucleotides and polypeptides of vascular endothelial growth factor 2 (VEGF-2) to treat disorders or damage to photoreceptor cells.

Technique. Related The formation of new blood vessels, or angiogenesis, is essential for embryonic development, KEF: 131066 subsequent growth and tissue repair. Angiogenesis is also an essential part of certain pathological conditions, such as neoplasia (ie, tumors and gliomas). Abnormal angiogenesis is associated with other diseases such as inflammation, rheumatoid arthritis, psoriasis and diabetic retinopathy (Folkman, J. and Klagsbrun, M., Science 235: 442-447 (1987)). Both molecules of the growth factor of the acidic and basic fibroblasts are mitogenic for endothelial cells and other cell types. Angiotropin and angiogenesis can induce angiogenesis, although their functions are not clear (Folkman, J., Cancer Medicine, Lea and Febiger Press, pp. 153-170 (1993)). A highly selective mitogen for vascular endothelial cells is the vascular endothelial growth factor or VEGF (Ferrara, N. et al., Endocr. Rev. 13: 19-32 (1992)), which is also known as vascular permeability factor (VPF). Vascular endothelial growth factor is a secreted angiogenic mitogen whose target cell specificity seems to be restricted to vascular endothelial 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 epithelial cells adjacent to the fenestrated endothelium, for example, in the choroid plexus and renal glomeruli. The data were consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation (Breier, G. et al., Developmen t 114: 521-532 (1992)). VEGF shares sequence homology with growth factors derived from human platelets, PDGFa and PDGFb (Leung, D.W., et al., Science 246: 1306-1309, (1989)). The degree of homology is approximately 21% and 23%, respectively. Eight cysteine residues that contribute to the formation of the disulfide bond are strictly observed in these proteins. Although they 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. Alternately spliced mRNAs have been identified for both VEGF, P GF, and PDGF and those different splicing products differ in biological activity and in receptor binding specificity. VEGF and PDGF function as homomers or heterodimers and bind to receptors which produce an intrinsic tyrosine kinase activity after receptor dimerization. VEGF has four different forms of 121, i 165, 189 and 206 amino acids due to alternative splicing. VEGF121 and VEGF165 are soluble and able to promote angiogenesis, while VEGF189 and VEGF206 bind to proteoglycans containing heparin on the cell surface. The temporal and spatial expression of VEGF has been correlated with the physiological proliferation of blood vessels (Gajdusek, C.M., and Carbon, S.J., Cell Physiol., 139: 570-579 (1989); McNeil, 'P. L., et al. , J. Cell. Biol. 109: 811-822 (1989)). Their high affinity binding sites are located only on endothelial cells in tissue sections (Jakeman, L.B., et al., Clin .. Invest. 89: 244-253 (1989)). The factor can be isolated from pituitary cells and from several tumor cell lines, 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 Chinese hamster ovary cells the ability to form tumors in nude mice (Ferrara, N. et al., J. Clin.Invest.91: 160-170 (1993 )). Inhibition of VEGF function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth in immunodeficient mice (Kim, K.J., Na ture 362: 841-844 (1993)). In addition, it was shown that a dominant negative mutant 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 to plasma proteins even after cessation of damage, which is a characteristic feature of normal wound healing. This suggests that VPF is an important factor in wound healing. Brown, L. F. et al. , J. Exp. Med. 1 76: 1375-1379 (1992). The expression of VEGF - is high in vascularized tissues, (for example, lung, heart, placenta and solid tumors) and correlates with both temporal and spatial angiogenesis. 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 to be used in the promotion of vascular tissue repair (for example in atherosclerosis) U.S. Patent No. 5,073,492, issued in December 17, 1991 to Chen et al. , describes a method for synergistically increasing the growth of endothelial cells in an appropriate environment comprising adding to the environment, VEGF, effectors and serum derived factor. Also, 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 a heterodimer or homodimer. The protein is a mitogen of mammalian vascular endothelial cells and, therefore, is useful for the promotion of vascular development and repair, as described in European Patent Application No. 92302750.2, published on September 30, 1992. Retina. The differentiated retina is composed of several types of cells: sensory (rods and conus photoreceptors), glia (Muller's cells), and two types of neurons, interneurons, (horizontal, bipolar, and ammacrine), and projection neurons (ganglion cells) The development of different types of cells in the retina does not occur in a synchronized manner with most cones, and the development of ganglionic and horizontal cells before birth. In contrast, the differentiation of a majority of the rods, the main cell type in the rat retina, occurs postnatally. Clonal analysis of progeny of retinal precursor cells has shown that these progenitor cells can produce various combinations of retinal cell types indicating that at least some of the progenitors are multipotential. In addition, findings from studies both in vivo and in vi tro suggest that the final phenotype of the cell is largely independent of the lineage, which suggests that the change of the microenvironment within the retina has a role in determining the cellular potential of the cell. the progenitor cells, as well as the differentiated phenotype of the progeny. In vi tro, the proliferation and differentiation of retinal cells is regulated by a variety of factors, eg, FGF-2, CNTF, LIF, TGF, retinoic acid, and BDNF, as well as by extracellular matrix and adhesion molecules cellular, for example s-laminin. Yang and Cepko (J. Neurosci.16 (19): 6089-6099 (1996)) and more recently Wen et al. (J. Biol. Chem. 273 (4): 2090-2097 (1998)) have identified and characterized the expression pattern of VEGFR-2 / FLK-1, a member of the VEGF receptor family. VEGFR transcripts are first detected in Eli.5 in association with the developing retinal vasculature and with the central region of the central retina (Yang and Cepko, J. Neurosci.16 (19): 6089-6099 (1996)) . Although it is not known whether the two events are related, this period of development is also marked by the beginning of the development of ganglion cells. At the day of the E15 development, the expression of VEGFR-2 extends to the periphery of the retina, consistent with the outward gradient of retinal development. The expression of VEGFR-2 was localized to a great extent in the ventricular zone during the perinatal period when neurogenesis is at its peak and a large number of postmitotic neurons are forming.

The PDGF / VEGF superfamily currently includes 7 members. The 5 members of the VEGF subfamily bind to 4 different VEGF tyrosine kinase receptors with different specificities, although superimposed. VEGF, a homodimeric glycoprotein of 34-36 kDa that is the member of the prototypic family, binds to VEGFR-1 and VEGFR-2. VEGF-B and VEGF-D bind only to VEGFR-1 or VEGFR-3, respectively. Although VEGF-C, VEGF-2, has the highest affinity for VEGFR-3, it also binds with a lower affinity to VEGFR-2. Once the VEGF receptors are activated, tyrosine phosphorylates a number of proteins downstream in the signal transduction pathway including phosphatidylinositol 3-kinase, phospholipase C, GAP, and Nck. Hereditary retinal degenerative diseases ("HRD diseases") are a group of inherited conditions in which progressive bilateral degeneration of the retinal structures leads to loss of retinal function; these diseases include, for example, age-related macular degeneration, a cause that leads to visual damage in the elderly; Leader's congenital amaurosis, which causes its victims to be born blind; and retinitis pigmentosa ("RP"). RP is the name given to those inherited retinopathies that are characterized by the loss of retinal photoreceptors (rods and cones), with retinal electrical responses to flashes of light (i.e. ---. * t »... ---. l ---- g ---? -. < you and. electroretinograms, or "ERG") that are reduced in amplitude. As the disease progresses, patients show attenuated retinal arterioles, and often show "spicular bony" pigmentation of the retina and waxy pallor of the optic discs. It is estimated that the incidence of RP in the United States is approximately 1: 3500 births. Familial cases of RP usually occur in childhood with night blindness and loss of the average peripheral visual field due to the loss of rods in the peripheral retina. As the condition progresses, the contraction of the visual fields eventually leads to blindness. Signs of fundus examination in advanced stages include attenuation of the retinal vessels, intraretinal pigment in the peripheral fundus, and waxy pallor of the optic disc. Patients have abnormal retinal electrical responses evoked by light (ie, electroretinograms or ERG), even in early stages in the absence of visible abnormalities in the examination of the fundus. Histopathological studies have revealed a widespread loss of photoreceptors in advanced stages. Therefore, there is a need in the art for a method for treating disorders and damage of photoreceptor cells.

Brief Description of the Invention The polypeptides of the present invention have been identified as a novel vascular endothelial growth factor based on the homology of the amino acid sequence with a human VEGF. In accordance with one aspect of the present invention, novel mature polypeptides, as well as biologically active fragments, analogues and derivatives thereof, are useful diagnostically or therapeutically. The polypeptides of the present invention are of human origin. According to another aspect of the present invention, isolated nucleic acid molecules are provided which comprise polynucleotides encoding full-length or truncated VEGF-2 polypeptides having the amino acid sequences shown in SEQ ID NOS: 2 or 4, respectively, or the amino acid sequence encoded by the cDNA clones deposited in bacterial hosts as Deposit Number ATCC 97149 on May 12, 1995 or ATCC Deposit Number 75698 on March 4, 1994. The present invention also relates to fragments , biologically active analogs and derivatives of VEGF-2 and useful diagnostically or therapeutically. According to yet another aspect of the present invention, processes for producing such polypeptides are provided by recombinant techniques, comprising i-a. -r a.- cultivate recombinant prokaryotic and / or eukaryotic host cells, containing a nucleic acid sequence coding for a polypeptide of the present invention, under conditions that promote the expression of proteins and the subsequent recovery of such proteins. with yet another aspect of the present invention, antibodies against such polypeptides and processes for producing such antibodies are provided. In accordance with another aspect of the present invention, nucleic acid probes are provided which comprise nucleic acid molecules of sufficient length to specifically hybridize to the nucleic acid sequences of the present invention. According to another aspect of the present invention, methods are provided for diagnosing diseases or a susceptibility to diseases related to mutations in nucleic acid sequences of the present invention and proteins encoded by such nucleic acid sequences. According to yet another aspect of the present invention, there is provided a process for using such polypeptides, or polynucleotides that encode | such polypeptides, for in vi tro purposes related to scientific research, DNA synthesis and manufacture of DNA vectors.

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According to still another aspect of the present invention, processes are provided for using such polypeptides or polynucleotides that encode such polypeptides for therapeutic purposes, for example, to protect or stimulate the growth of photoreceptor cells. According to yet another aspect of the present invention, antagonists of such polypeptides are provided, which can be used, for example, to inhibit or prevent the growth of photoreceptors, Those and other aspects of the present invention should be apparent to those experts in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1E show the full length nucleotide (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID NO: 2) of VEGF-2i. The polypeptide comprises approximately 419 residual amino acids of which approximately 23 represent the leader sequence. Abbreviations of a standard I letter for amino acids were used. The sequencing is performed using the Automated DNA Sequencer from Model'o 373 (Applied Biosystems, Inc.). It was described that the accuracy of the sequencing is greater than 97%.

Figures 2A-2D show the nucleotide (SEQ ID NO: 3) and the deduced amino acid sequence (SEQ ID p: 4) of a biologically active truncated form of VEGF-2. The polypeptide comprises approximately 350 residual amino acids of which approximately the first 24 amino acids represent the leader sequence. Figures 3A-3B are an illustration of the amino acid sequence homology of PDGFa (SEQ ID NO: 5), PDGFb (SEQ ID NO: 6), VEGF (SEQ ID NO: 7), and VEGF-2 (SEQ ID NO: 4). The boxed areas indicate the conserved sequences and the location of the eight conserved cysteine residues. Figure 4 shows the percent homology between PDGFa, PDGFb, VEGF, and VEGF-2. Figure 5 shows the presence of VEGF-2 mRNA in lines of human breast tumor cells. Figure 6 describes the results of a Northern electroblot analysis of VEGF-2 in human adult tissues. Figure 7 shows a photograph of a gel of SDS-PAGE after an in vitro trription, tration and electrophoresis of the polypeptide of the present invention; Lane 1: 14C and marker of P.M. rainbow; Lane 2: control of FGF; Lane 3: VEGF-2 produced by forward and forward M13 primers; Lane 4: VEGF-2 produced by reverse primers M13 and VEGF-F4; Lane 5: VEGF-2 produced by reverse primers M13 and VEGF-F5. Figures 8A and 8B describe photographs of SDS-PAGE gels. The VEGF-2 polypeptide was expressed in a baculovirus system consisting of Sf9 cells. The proteins of the medium and the cytoplasm of the cells were analyzed by SDS-PAGE under non-reductive (Figure 8A) and reducing (Figure 8B) conditions. Figure 9 depicts a photograph of an SDS-PAGE gel. The medium of Sf9 cells infected with a nucleic acid sequence of the present invention was precipitated. The resuspended pellet was analyzed by SDS-PAGE and stained with Coomasie brilliant blue. Figure 10 depicts a gel photograph of SDS-PAGE. VEGF-2 was purified from the supernatant of the medium and analyzed by SDS-PAGE in the presence or absence of the reducing agent b-mercaptoethanol and stained by bright blue Coomassie Figure 11 describes the reverse phase CLAP analysis of purified VEGF-2 using a RP-300 column (0.21 x 3 cm, Applied Biosystems, Inc.). The column was balanced with 0.1% of trifluoroacetic acid (Solvent A) and the proteins eluted with a gradient of 0 to 6% Solvent.

B for 7.5 min, acetonitrile compounds with a content of 0.07% TFA. The elution of the protein was verified by absorbance at 215 nm ("red" line) and 280 nm ("blue" line). The percentage of Solvent B is shown by the "green" line. Figure 12 shows a schematic representation of the expression vector pHE4-5 (SEQ ID NO: 9) and the cDNA of the subcloned VEGF-2 coding for the sequence, the gene locations marked for kanamycin resistance, the coding sequence of the VEGF-2 or the sequence or iC, and the coding sequence laclq are indicated. Figure 13 shows the nucleotide sequence of the regulatory elements of the pHE promoter (SEQ ID NO: 10). The two sequences of the lac operator, the Shine-Delgarno sequence (S / D), and the restriction sites Hindl I I and | Ndel terminals (in italics) are indicated. Figures 14A-D show that treatment with VEGF-2 increases the level of rhodopsin protein and the number of photoreceptor cells. The dissociated retinal cells were prepared from Pl animals, placed at a density of 425 cells / mm 2 and treated with VEGF-2 (A and B) or VEGF-2 (C and D). After 2, (open boxes), 5 (solid boxes), 7 (open circles), or 9 (solid squares) days, the total number of cells in the cultures was estimated by measuring the emission of calcein. The cultures were then fixed and the level of rhodopsin protein quantified by ELISA.

A-t- Figure 15 shows that the number of rhodopsin-immunopositive cells increased as a function of the VEGF-2 concentration. The retinal cells were maintained for 8 days in the presence of either VEGF-1 or VEGF-2. The cultures were then fixed and stained immunihistochemically for the determination of rhodopsin. Figure 16A-C shows that VEGF-2 increases the incorporation of BrdU and [3 H] thymidine in retinal cultures in a developmentally restricted manner. Cells from Pl animals were isolated and cultured at a density of 425 cells / mm 2. The cultures were initially treated for 4 hours after cultivation with VEGF or VEGF-2. After 1, 2, or 3 days, the cultures were labeled for 4 hours with BrdU. The cells were then fixed and processed by immunohistochemistry of BrdU. Figures 17A-B show the loss of response to VEGF-2 or VEGF-1 as a function of the time elapsed after isolation of the cells and the initial addition of the factors. Initially, a set of cultures with factors was treated 4 hours after the culture (9/0) and subsequently, additional sets were treated 24 or 48 hours later (8/1 or 7/2, respectively). After 5 days in culture, the cells were fixed and the rhodopsin prine level was quantified by ELISA assay.

Figures 18A-C show that VEGF increases the number of Amcrine cells but not Muller or Endothelial cells. The retinal cells were treated for 8 days with the indicated concentrations of VEGF-2. The cells were then fixed and stained immunohistochemically for the determination of syntaxin (A), analyzed, to determine the level of absorption of high affinity GABA (B), or GFAP (C). Figures 19A-C show the effect of developmental age on the response to VEGF-2. The retinal cells derived from animals E15 (A), E20 (B) or Pl (C) were cultured at a density of 212 (open squares), 318 (solid frames), or 425 cells / mm2. Four hours after the culture, the cultures were treated with the indicated concentrations of VEGF-2. 24 hours later, the cultures were changed to serum free medium and the factors were added again. The cultures were then labeled with [3H] thymidine 48 hours later. Figures 20A-B compare the response of retinal cells to VEGF-2 and other factors.) The cultures were seeded at a density of 425 cells / mm2 and treated for 9 days. Panel A shows that the total number of cells in the cultures was estimated using calcein, while panel B shows that the level of rhodopsin protein was determined by ELISA assay.

Figures 21A-C show that CNTF inhibits the response of photoreceptor cell progenitors to VEGF-2. The retinal cultures were treated 24 hours later by culturing with the indicated concentrations of CNTF in the presence or absence of 150 ng / ml of VEGF-2. 8 days later in vi tro, the amount of rhodopsin protein. was quantified (A) and the total number of cells in the cultures was determined (B). (C) To determine the effect of treatment with CNTF on the initial proliferative response induced by VEGF-1, the cultures were treated with the indicated concentrations of VEGF-2 in the presence or absence of 100 ng / ml of CNTF. 48 hours later, the cultures were labeled for 4 hours with [3 H] thymidine. Figure 22 shows the proliferation of improved ECL in response to treatment with VEGF2 and antibody. Figure 23 shows the proliferation of LEC in response to VEGF-2 and the combination of VEGF2: antibody. Figure 24 shows the map of the epitope for murine anti-VEGF-2 monoclonal antibodies. Figure 25 shows the status of the murine VEGF-2 monoclonal antibodies.

Detailed Description of the Preferred Modalities In accordance with one aspect of the present invention, isolated nucleic acid molecules comprising a polypeptide encoding a VEGF-2 polypeptide having an amino acid sequence deduced from Figure 1 (SEQ. ID NO: 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 Boule ard, Manassas, VA 20110-2209 , and to which ATCC Dept. No. 97149 was given. In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules comprising a polynucleotide encoding a truncated VEGF-2 polypeptide having the sequence of deduced amino acids 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 in the American Type Tissue Collection (ATCC), 10801 University Boulevard, Manassas. VA 20110-2209, and to which the Deposit Number i ATCC 75698 was given. Unless otherwise indicated, all! the nucleotide sequences determined by the ilento sequence ; ^ i ^ ^ ^, - ^ of a DNA molecule here were determined using an automated DNA sequencer (such as Model 373 from Applied Biosystems, Inc.). and all the amino acid sequences of the polypeptides encoded by the DNA molecules determined here were estimated by translating a DNA sequence determined as s, and did previously. Therefore, as is known in the art for any DNA sequence determined by this automated method, any nucleotide sequence | Here certain may contain some errors. The nucleotide sequences determined by automation are, typically, at least about 90% identical, more particularly at least about 95% up to | less approximately 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be determined more accurately by other methods including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a given nucleotide sequence compared to the actual sequence will produce a deviation of the frame in the translation i of the nucleotide sequence, so that the predicted amino acid sequence encoded by a nucleotide sequence determined will be completely different, to the amino acid sequence actually encoded by the sequenced DNA molecule, starting at the point of such insertion or deletion. A polynucleotide encoding a polypeptide of the present invention can be obtained from human embryo osteoblastomas at the initial stage (week 8 to 9), adult heart cell lines or various breast cancers. The polynucleotide of this invention was discovered in a cDNA library derived from human embryo in the | initial stage, week 9. This is structurally related to the VEGF / PDGF family. It contains an open reading frame that encodes a protein of approximately 419 residual amino acids of which approximately the first 23 residual amino acids are the putative leader sequence, so that the protein comprises 396 amino acids, and that the protein exhibits the sequence homology of higher amino acids with human vascular endothelial growth factor (30% identity), followed by PDGFa (24%) and PDGFb (22%). (See Figure 4). It is particularly important that the eight cysteines I are conserved within the four family members (see the boxed areas of Figure 3). In addition, the signature of the PDGF / VEGF family, PXCVXXXRCXGCCN, (S¡EQ ID NO: 8) is conserved in VEGF-2 (see Figure 31). The homology between VEGF-2, VEGF and the two PDGFs is at and level of the protein sequence. It can not be detected homology of the nucleotide sequence, and therefore, isolate VEGF-2 through simple methods such as a looser hybridization. It means that the VEGF-2 polypeptide of the present invention includes a full length polypeptide and the polynucleotide sequence coding for any leader sequences and for active fragments of the full length polypeptide. It means that > the active fragments include any portions of the full-length amino acid sequence that have rpenos of the total of 419 amino acids of the full-length amino acid sequence as shown in SEQ ID NO: 2, but still contain the eight cysteine residues shown preserved in FIGURE 3 and still having VEGF-2 activity. Exist . . at least two alternatively spliced VEGF-2 mRNA sequences present in normal cells. The two bands in FIGURE 7, lane 5 indicate the presence of alternately spliced mRNA that codifies for 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, which DNA includes cDNA, genomic DNA and synthetic DNA. DNA can be double-stranded or single-stranded, and if it is single stranded, it can be the coding strand or the non-coding strand (antisense). The coding sequence coding for the mature polypeptide may be identical to the coding sequence shown in Figure 1 or Figure 2, or to that of the deposited clones, or it may be a different coding sequence which, as a result of redundancy or degeneracy of the genetic code, it codes for the same mature polypeptide as the DNA of Figure 1, Figure 2, or the deposited cDNAs. The polynucleotide encoding the mature polypeptide of Figure 1 or Figure 2 or for the mature polypeptide encoded by the deposited cDNAs may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequences such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences, such as introns or non-coding sequences 5 'and / or 3' of the coding sequence of the mature polypeptide Thus, the term "polynucleotide! which encodes a polypeptide "encompasses a polynucleotide which includes only coding sequences for the polypeptide, as well as a polynucleotide which includes additional coding and / or non-coding sequences.

The present invention also relates to several of the polynucleotides described hereinabove. , which encode fragments, analogues and derivatives of polypeptides having the amino acid sequence deduced from Figures 1 or 2, or the polypeptide encoded by the cDNA of the deposited clones. The variant of the polynucleotide can be a natural allelic variant of the polynucleotide or a non-natural variant of the polynucleotide. Thus, the present invention -includes polynucleotides encoding the same mature polypeptide shown in Figures 1 or 2 or the same mature polypeptide encoded by the cDNA of the deposited clones, as well as variants of such polynucleotides, variants which code for a fragment, derivative or analog 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 a natural allelic variant of the coding sequence shown in Figures 1 or 2, or of the coding sequence of the deposited clones. As is known in the art, an allelic variant is an alternate form of a polynucleotide sequence having 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 mature polypeptide can be fused in the same reading frame to a polynucleotide that aids the expression and secretion of a polypeptide from a host cell), for example, a leader sequence that it 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 cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also code for a protein that is mature protein plus additional 5 'residual amino acids. A mature protein that has a prosequence is a protein and is an inactive form of the protein. Once the prosequence is cleaved, an active mature protein remains. Thus, for example, the polynucleotide I of the present invention can encode a mature protein, or for a protein having a prosequence or for a protein having a prosequence and a pre-sequence (leader sequence). The polynucleotides • - of the present invention may also have the coding sequence fused in frame to a marker sequence that allows purification of the polypeptide of the present invention. The marker sequence can be a hexahistidine tag supplied by the pQE-9 vector to provide for the purification of the mature polypeptide fused to the tag in the case of a bacterial host, or, for example, the tag sequence can be a tag of hemagglutinin (HA ) when a mammalian host is used, for example COS-7 cells The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, ii, et al., Cell 37: 1 61 (1984 ).) Additional embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having at least an 95% identical nucleotide sequence, and more preferably at least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence which codes for the polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence s of SEQ ID NO: 2, 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 396 in SEQ ID NO: ID NO: 2; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in the ATCC Deposit # 97149; (e) a nucleotide sequence that encodes! for the mature VEGF-2 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97149; or (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e). Additional 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 99% identical to (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO: 4; (b) a nucleotide sequence encoding the polypeptide having the amino acid sequence of 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: ID NO: 4; (d) a nucleotide sequence encoding the polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75698; (e) a nucleotide sequence encoding the mature VEGF-2 polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Depot No. 75698; or (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e). A polynucleotide having at least one nucleotide sequence, for example, 95% "identical" to a reference nucleotide sequence encoding a VEGF-2 polypeptide is that nucleotide sequence of the polynucleotide that is intended to be identical to the reference sequence except that the polynucleotide sequence may include up to five mutation points per hundred 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 total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5N or 3N terminal positions of the reference nucleotide sequence or anywhere between those terminal, interdispersed lentre positions - --- and --- -y.1 --- .y '. «£? --- ..- •' -. «-» - »» i- individual nucleotides in the reference sequence or in one or more contiguous individual groups within the reference sequence. As a practical aspect, 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 the nucleotide sequence of the deposited cDNA clones 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 ). The Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best homology segment between two sequences. When the 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 fixed, of course, so that the percentage of identity is calculated on the total length of the reference nucleotide sec- tion and that spaces are allowed in the homology of up to 5% of the total number of nucleotides in the reference sequence. Variants of VEGF-2 may contain alterations in the coding regions, non-coding regions or both. Especially preferred are polynucleotide variants which contain alterations which produce silent substitutions, additions or deletions, but which do not alter the properties or activities of the encoded polypeptide. The nucleotide varieties produced by silent substitutions due to the degeneracy of the genetic code are preferred. In addition, variants in which 5-10, 1-5, b 1-2 amino acids are substituted, deleted or added in any combination are also preferred. Variants of the VEGF-2 polynucleotide can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (codon change in human mRNA to those preferred by bacterial hosts such as E. coli). For example, changes directed to the site at the amino acid level of VEGF-2 can be made by replacing a particular amino acid with a conservative amino acid. Preferred conservative mutations include: Ml replaced with A, G, I, L, S, T, or V; H2 replaced with K, or R; S3 L4 replaced with A, G, I, S, T, M, or V; G5 replaced with A, I, L, S, T, M, or V; F6 replaced with W, or Y; F7 replaced with W, or Y; S8 replaced with A, G, I, L, T, M, or V; V9 replaced with A, G, I, L, S, T, or M; AlO replaced with G, I, L, S, T, M, or V; S12 replaced with A, G, I, L, T, M, or V; L13 replaced with A, G, I, S, T, M, or V; L14 replaced with A, G, I, S, T, M, or V; A15 replaced with G, I, L, S, T, M, or V; A16 replaced with G, I, L, S, T, M, or V; A17 replaced with G, I, L, S, T, M, or V; L18 replaced with A, G, I, S, T, M, or V; L19 replaced with A, G, I, S, T, M, or V; G21 replaced with A, I, L, S, T, M, or V; R23 replaced with H, or K; E24 replaced cpn D; A25 replaced with G, I, L, S, T, M, or V; A27 replaced with G, I, L, S, T, M, or V; A28 replaced with G, I, L, S, T, M, or V; A29 replaced with G, I, L, S, T, M, or V; A30 replaced with G, I, L, S, T, M, or V; A31 replaced with G, I, L, S, T, M, or V; F32 replaced with W, or Y; E33 replaced with D; S34 replaced with I A, G, I, L, T, M, or V; G35 replaced with A, I, L, S, T, M, or V; L36 replaced with A, G, I, S, T, M, or V; D37 replaced with E; L38 replaced with A, G, I, S, T, M, or V; S39 replaced with A, G, I, L, T, M, or V; D40 replaced with E; A41 replaced with G, I, L, S, T, M, or V; E42 replaced with D; D44 replaced with E; A45 replaced with G, I, L, S, T, M, or V; G46 replaced with A, I, L, S, T, M, or V; E47 replaced with D; A48 replaced with G, I, L, S, T, M, or V; T49 replaced with A, G, I, L, S, M, or V; A50 replaced with G, I, L, S, T, M, or V; Y51 replaced with F, or W; A52 replaced with G, I, L, S, T, M, or V; S53 replaced with A, G, I, L, T, M, or V; K54 replaced with H, or R; D55 replaced with E; L56 replaced with A, G, I, S, T, M, or V; E57 replaced with D; E58 replaced with D; Q59 replaced with N; L60 replaced with A, G, I, S, T, M, or V; R61 replaced with H, or K; S62 replaced with A, G, I, L, T, M, or V; V63 replaced with A, G, I, L, S, T, or M; S64 replaced with A, G, I, L, T, M, or V; S65 replaced with A, G, I, L, T, M, or V; V66 replaced with A, G, I, L, S, T, or M; D67 replaced with E; E68 replaced with D; L69 replaced with A, G, I, S, T, M, or V; M70 replaced with A, G, I, L, S, T, or V; T71 replaced with A, G, I, L, S, M, or V; V72 replaced with A, G, I, L, S, T, or M; L73 replaced with A, G, I, S, T, M, or V; Y74 replaced with F, or W; E76 replaced with D; Y77 replaced with F, or W; W78 replaced with F, or Y; K79 replaced with H, or R; M80 replaced with A, G, I, L, S, T, or V; Y81 replaced with F, or W; K82 replaced with H, or R; Q84 replaced with N; L85 replaced with A, G, I, S, T, M, or V; R86 replaced with H, or K; K87 replaced with H, or R; G88 replaced with A, I, L, S, T, M, or V; G89 replaced with A, I, L, S, T, M, or V; W90 replaced with F, or Y; Q91 replaced with N; H92 replaced with K, or R N93 replaced with Q; R94 replaced with H, or K E95 replaced with D; Q96 replaced with N; A97 replaced with G, I, L, S, T, M, or V; N98 replaced with Q; L99 replaced with A, G, I, S, T, M, or V; N100 replaced with Q; S101 replaced with A, G, I, L, T, M, or V; R102 replaced with H, or K; T103 replaced with A, G, I, ¡L, S, M, or V; E104 replaced with D; E105 replaced with D T106 replaced with A, G, I, L, S, M, or V; 1107 replaced with A, G, L, S, T, M, or V; K108 replaced with H, or R; F109 replaced with W, or Y; A110 replaced with G, I, L, S, T, M, or V; There replaced with G, I, L, S T, M, or V; A112 replaced with G, I, L, S, T, M, or V; HI13 replaced with K, or R; Y114 replaced with F, or W; N115 replaced with Q; T116 replaced with A, G, I, L, S, M, or V; E117 replaced with D; 1118 replaced with A, G, L, S, T, M, or V; L119 replaced with A, G, I, S, T, M, or V; K120 replaced with H, or R; S121 replaced with A, G, I, L, T, M, or V; 1122 replaced with A, G, L, S, T, M, or V; D123 replaced with E; N124 replaced with Q; E125 replaced with D; W126 replaced with F, or Y; R127 replaced with H, or K; K128 replaced with H, or R; T129 replaced with A, G, I, L, S, M, or V; Q130 replaced with N; M132 replaced with A, G, I, L, S, T, or V; R134 replaced with H, or K; E135 replaced with D; V136 A, G, I, L, T, M, or V; K183 replaced with H, or R; T184 replaced with A, G, I, L, S, M, or V; L185 replaced with A, G, I, S, T, M, or V; F186 replaced with W, or Y; E187 replaced with D; T188 replaced with A, G, L, S, T, | M, V; T189 replaced with A, G, I, L, S, M, or V; V190 replaced with A, G, I, L, S, T, or M; L192 replaced c; on A, G, I, S, T, M, or V; S193 replaced with A, G, I, L, T, M, or V; Q194 replaced with N; G195 replaced with A, I, L, S, T, M, or V; K197 replaced with H, or R; V199 replaced with A, G, I, L, S, T, or M; T200 replaced with A, G, I, L, S, M, or V; 1201 replaced with A, G, L, S, T, M, or V; S202 replaced with A, G, I, L, T, M, or V; F203 replaced with W, or Y; A204 replaced with G, I, L, S, T, M, or V; N205 replaced with Q; H206 replaced with K, or R; T207 replaced with A, G, I, L, S, M, or V; S208 replaced with A, G, I, L, T, M, or V; R210 replaced with H, or K; M212 replaced with A, G, I, L, S, T, or V; S213 replaced with A, G, I, L, T, M, or V; K214 replaced with H, or R; L215 replaced with A, G, I, S, T, M, or V; D216 replaced with E; V217 replaced with A, G, I, L, S, T, or M; Y218 replaced with F, or W; R219 replaced with H, or K; Q220 replaced with N; V221 replaced with A, G, I, L, S, T, or M; H222 replaced with K, or R; S223 replaced with A, G, I, L, T, M, or V; 1224 replaced with A, G, L, S, T, M, or V; 1225 replaced with A, G,, S, * ^ «Faith * ^^^^^^« ^^^ i ^^ T, M, or V; R226 replaced with H, or K; R227 replaced with H, or K; S228 replaced with A, G, I, L, T, M, or V; L229 replaced with A, G, I, S, T, M, or V; A231 replaced with G, I, L, S, T, M, or V; T232 replaced with A, G, I, L, S, M, or V; L233 replaced with A, G, I, S, T, M, or V; Q235 replaced with N; Q237 replaced with N; A238 replaced with G, I, L, S, T, M, or V; A239 replaced with G, I, L, S, T, M, or V; N240 replaced with Q; K241 replaced with H, or R; T242 replaced with A, G, I, L, S, M, V; T245 replaced with A, G, I, L, S, M, or V; N246 replaced with Q; Y247 replaced with F, or W; M248 replaced with A, G, I, L, S, T, or V; W249 replaced with F, or Y; N250 replaced with Q; N251 replaced with Q; H252 replaced with KJ or R; 1253 replaced with A, G, L, S, T, M, or V; R255 replaced with H, or K; L257 replaced with A, G, I, S, T, M, or V; A258 replaced with G, I, L, S, T, M, or V; Q259 replaced with N; E260 replaced with D; D261 replaced with E; F262 replaced with W, or Y; M263 replaced with A, G, I, L, S, T, or V; F264 replaced with W, or Y; S265 replaced with A, G, I, L, T, M, or V; S266 replaced with A, G, I, L, T, M, or V; D267 replaced with E; A268 replaced with G, I, L, S, T, M, or V; G269 replaced with A, I, L, S, T, M, or V; D270 replaced with E; D271 replaced with E; S272 replaced with A, G, I, L, T, M, or; T273 replaced with A, G, I, ¡L, S, M, or V; D274 replaced with E; G275 replaced with A, I, L, S, T, M, or V; F276 replaced with W, or Y; H277 replaced with K, or R; D278 replaced with E; 1279 replaced with A, G, L, S, T, M, or V; G281 replaced with A, I, L, S, T, M, or V; N283 replaced with Q; K284 replaced with H, or R; E285 replaced with D; L286 replaced with A, G, I, S, T, M, or V; D287 replaced with E; E288 replaced with D; E289 replaced with D; T290 replaced with A, G, I, L, Sj M, or V; Q292 replaced with N; V294 replaced with A, G, I, L, S, T, or M; R296 replaced with H, or K; A297 replaced with G, I, L, S, T, M, or V; G298 replaced with A, I, L, S, T, M, or V; L299 replaced with A, G, I, S, T, M, or V; R300 replaced with H, or K; A302 replaced with G, I, L, S, T, M, or V; S303 replaced with A, G, I, L, T, M, or V; G305 K; E333 replaced with D; F334 replaced with W, or Y; D335 replaced with E; E336 replaced with D; N337 replaced with Q; T338 replaced with A, G, I, L, S, M, or V; Q340 replaced with N; V342 replaced with A, G, I, L, S, T, or M; K344 replaced with H, or R; R345 replaced with H, or K; T346 replaced with A, G, I, L, S, M, or V; R349 replaced with H, or K; N350 replaced with Q; Q351 replaced with N; L353 replaced with A, G, I, S, T, M, or V; N354 replaced with Q; G356 replaced with A, I, L, S, T, M, or V; K357 replaced with H, or R; A359 replaced with G, I, L, S, T, M, or V; E361 replaced with D; T363 replaced with A, G, I, L, S, M, or V; E364 replaced with D; S365 replaced with A, G, I, L, T, M, or V; Q367 replaced with N; K368 replaced with H, or R; L370 replaced with A, G, I, S, T, M, or V; L371 replaced with A, G, I, S, T, M, or V; K372 replaced with H, or R; G373 replaced with A, I, L, S, T, M, or V; K374 replaced with H, or R; K375 replaced with H, or R; F376 replaced with W, or Y; H377 replaced with K, or R; H378 replaced with K, or R; Q379 replaced with N; T380 replaced with A, G, I, L, S, M, or V; S382 replaced with A, G, I, L, T, M, or V; Y384 replaced with F, or W; R385 replaced with H, or K; R386 replaced with H, or K; T389 replaced with A, G, I, L, S, M, or V; N390 replaced with Q; R391 replaced with H, or K; Q392 replaced with N; K393 replaced with H, or R; A394 replaced with G, I, L, S, T, M, or V; E396 replaced with D; G398 replaced with A, I, L, S, T, M, or V; F399 replaced with W, or Y; S400 replaced with A, G, I, L, T, M, or V; Y401 replaced with F, or W; S402 replaced with A, G, I, L, T, M, or V; E403 replaced with D; E404 replaced with D; V405 replaced with A, G, I, L, S, T, or M; R407 replaced with H, or K; V409 replaced with A, G, I, L, S, T, or M; S411 replaced with A, G, I, L, T, M, or V; Y412 replaced with F, or W; W413 replaced with F, or Y; Q414 replaced with N; R415 replaced with H, or K; Q417 replaced with N; M418 replaced with A, G, I, L, S, T, or V; and / or S419 replaced with A, G, I, L, T, M, or V of Figure 1. The resulting constructs can be routinely separated by their activities or functions described through the specification and known in the art. . Preferably, the resulting constructs have an increased and / or decreased VEGF-2 activity, while the remaining VEGF-2 activities or functions are maintained. More preferably, the resulting constructs have increased or decreased VEGF-2 activity or function, while activities o. remaining VEGF-2 functions are maintained. In addition to the substitution of conservative amino acids, variants of VEGF-2 include (i) substitutions with one or more of the non-conserved residual amino acids, where the substituted residual amino acids may or may not be one encoded by the genetic code, or (ii) substitution with one or more residual amino acids having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and / or solubility of the polypeptide (eg, polyethylene glycol), or (iv) ) fusing the polypeptide with additional amino acids, such as, for example, a peptide from the Fc region of IgG, or leader or secretory sequence, or a sequence facilitating purification. It is considered that such variant polypeptides are within the scope of those skilled in the art from the teachings herein. For example, the polypeptide variants of the VEGF-2 containing amino acid substitutions of amino acids loaded with other charged or neutral amino acids can produce proteins with improved characteristics, such as less aggregation. The aggregation of pharmaceutical formulations reduces the activity and increases the elimination due to the immunogenic activity of the aggregate. (Pincka rd et al., Clin. Exp. Immunol., 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems. 10: 307-377 (1993)).

For example, preferred non-conservative VEGE-2 substitutions include: MI replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H2 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S3 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L4 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F6 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F7 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, d C; S8 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V9 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; AlO replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Cll replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S12 replaced with D, E, H, K, R, N, Q, F, W, Y, p, C; L13 replaced with D E, H, K, R, N, Q, F, W, Y, P, or C; L14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A15 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; At 6 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A17 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L18 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L19 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P20 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G21 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P22 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, w | And, or C; R23 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E24 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P26 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A27 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A30 replaced with D, E, H, K, R, N, Q, F, W, Y, or C; A31 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F32 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; E33 replaced with H, K, R, N, Q, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S34 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G35 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D37 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D40 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E42 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P43 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D44 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G46 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E47 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A48 replaced with - »s -« # * • # &g; & p D, E, H, K, R, N, Q, F, W, Y, P, or C; T49 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y51 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S53 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K54 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D55 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E57 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E58 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q59 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L60 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R61 replaced with D, E, A, G, I, L, S, T,, M, V, N, Q, F, W, Y, P, or C; S62 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C • S65 replaced with D, E, H, K, R, N, Q, F, W, Y , P, or C; V66 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D67 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E68 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M70 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T71 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V72 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L73 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y74 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; «---.

P75 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E76 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y77 replaced cor. D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; W78 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K79 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M80 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y81 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K82 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C83 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q84 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L85 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R86 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K87 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G88 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G89 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W90 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q91 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y ,. P, or C; H92 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N93 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R94 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E95 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q96 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N98 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L99 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N100 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S101 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R102 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T103 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E104 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E105 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 1107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K108 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F109 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; There replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H113 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y114 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N115 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T116 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E117 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 1118 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K120 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S121 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 1122 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D123 replaced with ¡H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N124 replaced with - t - ^^ éte »- replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A147 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N149 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T150 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F151 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F152 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K153 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P 154 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P155 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C156 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V157 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S158 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V159 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y160 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R161 replaced with D, E, A, G, I, L, S, T, M, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S168 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E169 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, O C; G170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L171 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q172 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C173 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; M174 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N175 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T176 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S177 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T178 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S179 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y180 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S182 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K183 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T184 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F186 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E187 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 1188 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T189 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P191 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, C; L192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C S193 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q194 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G195 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P196 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K197 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P198 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 1201 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S202 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F203 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N205 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H206 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S208 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C209 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R210 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C211 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; M212 replaced with D, E, H, K, Q235 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C236 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q237 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A238 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A239 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N240 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K241 replaced with D, E, A, G, I, L, ¿, T, M, V, N, Q, F, W, Y, P, or C; T242 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C243 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P244 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T245 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N246 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Y247 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; M248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W249 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N250 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N251 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H252 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 1253 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C254 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R255 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C256 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L257 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q259 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E260 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D261 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F262 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; M263 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F264 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S265 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D267 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A268 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D270 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D271 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S272 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T273 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D274 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G275 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F276 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H277 replaced with D, E, A, G, I, L, S, T, M, .ji -.-. t - -3 ------- j ---- t -. it-- V, N, Q, F, W, Y, P, or C; D278 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 1279 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C280 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P282 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N283 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K284 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E285 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L286 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D287 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E288 replaced with H, K, R, A, G, I,, S, T, M, V, N, Q, F, W, Y, P, or C; E289 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T290 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C291 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q292 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C293 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V294 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C295 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, f., Q, F, W, Y, or P; R296 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A297 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G298 replaced with D, E, ifct ^^? ^ tA¿á SW? j »H, K, R, N, Q, F, W, Y, P, or C; L299 replaced with D E, H, K, R, N, Q, F, W, Y, P, or C; R300 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C P301 replaced with D, E, H, K, R, A, G , I, L, S, T, M, V, N, Q, F, W, Y, or C; A302 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S303 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C304 replaced with D, E, H, K, R, A, G, | I, L, S, T, M, V, N, Q, F, W, Y, or P; G305 replaced with D, | E, H, K, R, N, Q, F, W, Y, P, or C; P306 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; H307 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K308 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E309 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L310 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D311 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R312 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N313 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S314 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C315 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q316 replaced with L, S, T, M, V, N, Q, F, W, Y, or P; K320 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, O C; N321 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K322 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L323 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F324 replaced with D E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P325 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S326 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q327 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C328 replaced with D, E, H,, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G329 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A330 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N331 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R332 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E333 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F334 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D335 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E336 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N337 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T338 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C339 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Q340 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C341 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V342 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C343 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K344 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R345 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T346 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C347 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P348 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R349 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N350 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Q351 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P352 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L353 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N354 replaced with D, E, H, K, R, A, G, I,, S, T, M, V, F, W, Y, P, or C; P355 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G356 replaced with D, E, H, K, R, N, Q. F, W, Y, P, or C; K357 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C358 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A359 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C360 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; E361 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C362 replaced with ¡D, E, b & H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; T363 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E364 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S365 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P366 replaced with D, E ,, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q367 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K368 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C369 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; L370 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L371 replaced with D, E, H, K, R, N, Q, F, W, Y, P or C; K372 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G373 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K374 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K375 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F376 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H377 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H378 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q379 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T380 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C381 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S382 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C383 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y384 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R385 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R386 replaced with D, E, A, G, I, L, S, T, g ^^^^^^ ^ ^ g ^^^^^^^^ M, V, N, Q, F, W, Y, P, or C; P387 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C388 reepplied with D, p, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; T389 reepplied with D, E, H, K, R, N, Q, F, W, Y, P, or C; N390 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, ', Y, P, or C; R391 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q392 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K393 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A394 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C395 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; E396 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P397 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G398 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F399 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S400 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y401 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S402 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E403 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,?, P, O C; E404 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V405 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C406 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R407 replaced with D, E, A, G, I, L, S, T, of the VEGF-2 protein. In addition, such polypeptides can be used in the yeast bihibited system to "capture" VEGF-2 protein binding proteins that are also candidate agonists and antagonists according to the present invention. Bihibited yeast systems are described in Fields and Song, Nature 340: 245-246 (1989). In another aspect, the invention provides a peptide or polypeptide comprising a portion that contains an epitope of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. An "immunogenic epitope" is defined as part of a protein that produces an antibody response when the entire protein is the immunogen. It is believed that these immunogenic epitopes will be confined to a few sites in the molecule. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope". The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al. , Proc. Nati Acad. Sci. USA 81: 3998-4002 (1983). For the selection of peptides or polypeptides containing an antigenic epitope (i.e., containing a region of a protein molecule to which an antibody can bind) it is well known in the art that relatively short synthetic peptides that mimic part of a Protein sequence are commonly capable of producing an antiserum that reacts with the partially imitated protein. See, for example Sutcliffe, J. G. et al. , (1983) Science 219: 660-666. Peptides capable of producing protein reactive serum are often represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are not confined to immunodominant regions of intact protein (ie, immunogenic epitopes) or to the amino or carboxy terminals. Peptides that are extremely hydrophobic and those with six or less residues are generally not effective in inducing the antibodies to bind to the mimicked protein; The larger soluble peptides, especially those containing proline residues, are usually effective. Sutcliffe et al. , supra, at 661. For example, 18 of the 20 peptides designated according to these guidelines, containing 8-39 residues that cover 75% of the polypeptide chain sequence of HAI influenza virus haemagglutinin, induced antibodies that reacted with the HAI protein or the intact virus; and 12/12 peptides of MuLV polymerase and 18/18 of rabies glycoprotein induced antibodies that precipitated the respective proteins. The peptides and polypeptides containing antigenic epitopes of the invention are therefore useful for producing antibodies, including monoclonal antibodies, that specifically bind to a polypeptide of the invention. In this way, a high proportion of the hybridomas obtained approximately 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a major 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 entire amino acid sequence of a polypeptide of the invention, are also considered peptides or polypeptides containing epitopes of the invention and are also useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the peptide containing the epitope is selected to provide substantial solubility in aqueous solvents (ie, that 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 antigenic polypeptides or peptides that can be used to generate specific antibodies to VEGF-2 incluyer. the following: a polypeptide comprising the residual amino acids from about leu-37 to about Glu45 in SEQ ID NO: 2, from approximately Tyr-5Í to approximately Gly-66 in SEQ ID NO: 2, from - ^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^ approximately Gln-73 to approximately Glu-81 in SEQ ID NO : 2, from about Asp-100 to about Cys-108 in SEQ ID NO: 2, from about Gly-140 to about Leu-148 in SEQ ID NO: 2, from about Pro-168 to about Val-176 in SEQ ID NO: 2, from about His-183 to about Lys-191 in 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 SEQ ID NO: 2, from about Asp-244 to about His-254 in SEQ ID NO: 2, from about Gly-258 to about Glu-266 in SEQ ID NO: 2, from about Cys -272 to about Ser-280 in SEQ ID NO: ID NO: 2, from about Pro-283 to about Ser-291 in SEQ ID NO: 2, from about Cys-296 to about Gln-304 in the SEQ ID NO: 2, from about Ala-307 to about Cys-316 in SEQ ID NO: 2, from about Val-319 to about Cys-335 in the SEQ ID NO: 2, from about Cys-339 to about Leu-347 in SEQ ID NO: 2, from about Cys-360 to about Glu-373 in the SEQ ID NO: 2, from about Tyr-37i to about Val-386 in SEQ ID NO: 2, from about Ser-388 to about Ser-396 in SEQ ID NO: 2. It has been determined that those polypeptide fragments contain epitopes Antigens of the VEGF-2 protein by the analysis of the Jameson-Wolf antigenic index. Peptides and polypeptides containing epitopes of the invention can be produced by any conventional p.dope to label peptides or polypeptides, including recombinant means, using nucleic acid molecules of the invention. For example, an epitope-containing amino acid sequence, cut, 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 containing epitopes can also be synthesized using known chemical synthesis methods. For example, Houghten has described a simple method for the synthesis of large numbers of peptides, such as 10-20 mg of 248 peptides from 13 different residues representing unique amino acid variants of a segment of the HAI polypeptide, which were prepared and characterized (by binding studies of the ELISA type) in less than four weeks. Houghten, R. A. (1985) G Proc. Na ti. Acad. Sci. USA 82: 5131-5135. This process of "Simultaneous Multiple Peptide Synthesis (SMPS)" is best described in the ^ ¿Mgjf ^^^ Animals such as rabbits, rats and mice are immunized with 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, for example, intervals of about 2 weeks, may be necessary to provide a useful titre of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to 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 according to well-known methods. The technique. Peptides containing immunogenic epitopes of the invention, ie, those portions of a protein that produce an antibody response when the entire protein is the immunogen, are identified according to methods known in the art. For example, Geysen et al. , supra, describes a procedure for rapid concurrent synthesis on solid sophores 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 removing them from the support. In this way a peptide containing an immunogenic epitope of a desired protein can be routinely identified by one skilled in the art. For example, the immunologically important epitope in the coat protein of the foot and mouth disease virus was localized by Geysen et al. with a resolution of 7 amino acids by synthesis of an overlapping set of all 208 possible hexapeptides that cover the entire 213 amino acid sequence of the protein. Next, a set of complete replacement peptides was synthesized in which all 20 amino acids were substituted at the same time at each position within the epitope, and the particular amino acids that confer specificity for the reaction with the antibody were determined. Thus, peptide analogs of the epitope-containing peptides of the invention can be produced routinely by this method. U.S. Patent No. 4,708,781 to Geysen (1987) further discloses this method for identifying a peptide that contains an immunogenic epitope of a desired protein. Moreover, U.S. Patent No. 5,194,392 to Geysen (1990) discloses a general method for detecting or determining the sequence of monomers (amino acids or other compounds) that is a topological equivalent of the epitope (ie, an Amimotope) that is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) discloses a method for detecting or determining a monomer sequence that is a topographic equivalent of a ligand that is complementary to the ligand binding site of a particular receptor. of interest. Similarly, U.S. Patent No. 5,480,971 to Houghten, R. A. et al. (1996) in Peralkylated Oligopeptide Mixtures disclose linear peralkylated C alqu-C7 alkyl oligopeptides and sets and libraries of such peptides, as well as methods for using such sets and oligopeptide libraries to determine the sequence of a peralkylated oligopeptide that preferably binds to a receptor molecule of interest. Thus, the non-peptidic analogs and the epitope-containing peptides of the invention can also be produced routinely by these methods. As one of ordinary skill in the art will appreciate, the VEGF-2 polypeptides of the present invention and the epitope-containing fragments thereof described above can be combined with portions of the constant domain of immunoglobulins (IgG), resulting in chimeric peptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, for example, for chimeric proteins ^^ j ^ s consisting of the first two domains of the human CD4 polypeptide and several domains of the constant regions of the heavy and light chains of mammalian immunoglobulin (EPA 394,827; Traunecker et al., Na ture 331: 84-86 ( 1988)). In accordance with the present invention, novel variants of VEGF-2 are also described. These can be produced by deleting or substituting one or more amino acids of VEGF-2. Natural mutations are called allelic variations. Allelic variations can be silent (without change in the purified polypeptide) or they can have altered amino acid sequences. To try to improve or alter the characteristics of native VEGF-2, the design of proteins can be used. The recombinant DNA technology can be used by those skilled in the art to create novel polypeptides. Muteins and deletions may show, for example, greater activity or greater stability. In addition, they could be purified with a high yield and show better solubility, at least under certain conditions of purification and storage. In the examples below, mutations that can be constructed are exposed.

Terminal amino and carboxy terminal deletions In addition, VEGF-2 appears to be proteolytically cleaved after expression, resulting in Ala (residue 24) to Ser (residue 419); Pro (25) a Ser (419); Wing (26) to Ser (419); Wing (27) to Ser (419); To (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) a Ser (419); Leu (37) to (Ser (419); Ser (38) to Ser (419); Asp (39) to Ser (419); Wing (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 i; Tyr (50) to Ser (419); Ser (52) to Ser (419); Asp (54) to Ser (419); Val (62) to Ser (419); Val (65) to Ser (419; Met (1 Glu (23), or Ala (24) to Met (418); Met (1), Glu (23), or Ala (24) to Gln (417); Met (1), Glu (23), or Ala (24) to Pro (416; Met i), Glu (23), or Ala (24) to Arg (415); Met (1), Glu (23), or Ala (24) to Gln (414; Met Glu (23; or Ala (24 to Trp (413); Met (1), Glu (23), or Ala (24) to Tyr (412); Met (1) Glu (23), or Ala (24) to Ser (411); Met (1), Glu (23), or Ala (24) to Pro (410); Met (1, Glu (23), or Ala (24) to Val; 409); Met (1), Glu (23), or Ala (24) to Cys (408); Met (1), Glu (23), or Ala (24) to Arg (407); Met (1), Glu (23), or Ala (24) to Cys (406); Met (1), Glu (23), or Ala (24) to Val (405); Met (1) Glu (23), or Ala (24) to Glu (404); Met (1), Glu (23), or Ala (24) to Glu (403); Met (1), Glu (23), or Ala (24) to Ser (402); Met (1), Glu (23), or Ala (24) to Gly (398); Met (1), Glu! 2. 3 ) , terminal and C-terminal deletion mutants described above. Those combinations can be made using recombinant techniques known to those skilled in the art. Particularly, the 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 residual amino acid identified in SEQ ID NO. : 2. Preferably, the N-terminal deletions retain the conserved boxed area of Figure 3 (PXCVXXXRCXGCCN) (SEQ ID NO: 8), and include polypeptides comprising the amino acid sequence of the residues: 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-396; G-12 to S-396; ? -13 to S396; D-14 to S-396; L-15 to S-396; S-16 to S-396; D-17 to S-396; A-18 to S396; E-19 to S-396; P-20 to S-396; D-21 to S-396; A-22 to S-396; G-23 to S396; E-24 to S-396; A-25 to S-396; T-26 to S-396; A-27 to S-396; Y-28 to S396; A-29 to S-396; S-30 to S-396; K-31 to S-396; D-32 to S-396; L-33 to S396; E-34 to S-396; E-35 to S-396; Q-36 to S-396; L-37 to S-3S6; R-38 to S396; S-39 to S-396; V-40 to S-396; S-41 to S-396; S-42 to S-396; V-43 to S396; D-44 to S-396; E-45 to S-396; L-46 to S-396; M-47 to S-396; T-48 to S396; V-49 to S-396; L-50 to S-396; Y-51 to S-396; P-52 to S-396; E-53 to S396; Y-54 to S-396; W-55 to S-396; K-56 to S-396; M-57 to S-396; Y-58 to S-396; K-59 to S-396; C-60 to S-396; Q-61 to S-396; L-62 to S-396; R-63 to S-396; K-64 to S-396; G-65 to S-396; G-6 6 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 S396; A-89 to S-396; H-90 to S-396; Y-91 to S-396; N-92 to S-396; T-93 to S396; E-94 to S-396; 1-95 to S-396; L-96 to S-396; K-97 to S-396; S-98 to S396; 1-99 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 S396; V-113 to S-396; C-114 to S-396; 1-115 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; P131 to S-396 of SEQ ID NO: 2. Polynucleotides encoding these N-terminal deletion mutants are also preferred. In addition, the C-terminal deletions of the VEGF-2 polypeptide can also be described by the general formula -23-n, where n is an integer from -15 to 395 where n corresponds to the position of the residual amino acid identified in SEQ ID. NO: 2. Preferably, the The C-terminal deletions retain the conserved boxed area of Figure 3 (PXCVXXXRCXGCCN) (SEQ ID NO: include the polypeptides that comprise the amino acid sequence of the residues: The a-M-395; 394; El a P-393; El a R-392; El a Q-391; El a W-390; El a Y-389; El a S-388; El a P-387; El a V-386; A to C-385; A to R-384; A to C-383; A to V-382; A to E-381; A to E-380; A to S-379; A to Y-378; S-377; El to F-376; El to G-375; El to P-374; El to E-373; El to C-372; El to A 371; El to K-370; El to Q-369;; A to R-368; El to N-367; El to T-366; El to C-365; El to P-364; El to R-363; El to R -362; El to Y-361; to C-360; El to S-359; El to C-358; El to T-357; El to Q-356; El to H-355; El to H-354; El to F-353; El to K -352; El to K-351; El to G350; El to K-349; El to L-348; El to L-347; El to C-346; El to K-345; El to Q-344; El to P-343; El to S-342; El to E-341; El to T-340; El to C-339; El to E-338; El to C-337; El to A-336; El to C -3 35; E-1 to K-334; E-l to G-333; E-l to P-332; E-l to N-331; E-1 to L-330; E-l to P-329; E-1 to Q-328; E-l to N-327; E-1 to R-326; E-l to P-325; E-1 to C-324; E-l to T-323; E-1 to R-322; E-l to K-321; E-l to C-320; E-l to V-319; E-1 to C-318; E-l to 317; E-l to C-316; E-l to T-315; E-l to N-314; E-l to E-313; The A D-312; E-l to F-311; E-l to E-310; E-1 to R-309; E-l to N-30; E-1 to A-307; E-l to G-306; E-1 to C-305; E-l to Q-304; E-1 to S-303; E-l to P-302; E-l to F-301; E-l to L-300; The A ? 299; E-l to N-298; E-l to K-297; E-1 to C-296; E-l to V-295; The A C-294; E-l to Q-293; E-1 to C-292; E-1 to S-291; E-l to N-290; E-1 to R-289; E-1 to D-288; E-l to L-287; E-l to E-286; The A K-285; E-1 to H-284; E-l to P-283; E-1 to G-282; E-1 to C-281; E-1 S-280; E-1 A-279; E-1 P-278; E-1 to R-277; He L-276; E-l 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-l to C268; E-l to T-267; E-l to E-266; E-l to E-265; E-1 to D-264; E-1 to L-263; The to E-262; E-l to K-261; E-l to N-260; E-l to P-259; E-l to G-258; E-l to C257; E-1 to 1-256; E-l to D-255; E-l to H-254; E-l to F-253; E-l to G-252; The a 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 G246; E-l to A-245; E-1 to D-244; E-1 to S-243; E-1 to S-242; E-l to F-241; The a M-240; E-l to F-239; E-1 to D-238; E-l to E-237; E-l to Q-236; E-l to A-235; E-l to L-234; E-1 to C-233; E-1 to R-232; E-1 to C-231; E-1 to 1-230; E-l to H-229; E-l to N-228; E-l to N-227; E-l to W-226; E-l to M-225; E-l to Y-224; E-l to N-223; E-l to T-222; E-l to P-221; E-l to C-220; E-l to T219; E-l to K-218; E-l to N-217; E-l to A-216; E-l to A-215; E-l to Q-214; E-1 to C-213; E-l to Q-212; E-l to P-211; E-l to L-210; E-l to T-209; E-l to A-208; E-l to P-207; E-l to L-206; E-1 to S-205; E-1 to R-204; E-1 to R-203; E-1 to 1-202; E-1 to 1-201; E-l to S-200; E-1 to H-199; E-1 to V-198; E-l to Q-197; E-1 to R-196; E-l to Y-195; E-l to V-194; E-1 to D-193; E-l to L-192; E-l to K-191; E-1 to S-190; E-l to M-189; E-1 to C-1 E-1 to R-187; E-1 to C-186; E-1 to S-185; E-l to T-184; E-l to H-183; E-l to N-182; E-l to A-181; E-l to F-180; E-1 to S-179; E-1 to 1-178; E-l to T-ip7; E- 1 to V-176; E-l to P-175; E-l to K-174; E-l to P-173; E-l 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-l to T-166; E-1 to 1-165; E-l to E-164; E-1 to F-163; E-1 to L-162; E-l to T-161; E-l to K-160; E-1 to S-159; E-l to L-158; E-l to Y-157; E-1 to S-156; E-l to T-155; E- 1 to S-154; E-l to T-153; E-l to N-152; E-l to M-151; E-1 to C-150; E-l to Q-149; E-l to L-148; E-l to G-147; E-l to E-146; E-1 to S-145; E-l to N-144; of SEQ ID NO: 2. Polynucleotides encoding these C-terminal deletion mutants are also preferred. Preferably, any of the N- or C-terminal deletions listed above can be combined to produce a N- and C-terminal deleted VEGF-2 polypeptide, which retains the conserved boxed domain. In addition, the invention also provides polypeptides; having one or more amino acids selected from both amino and carboxy termini, which can be described generally as having m-n residues of SEQ ID NO: 2, where n and m are integers as described above. Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of those sequences are related to SEQ ID NO: 1 and may have become 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 embarrassing. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula ab, wherein a is an integer from 1 to 1760 of SEQ ID NO: 1, b is an integer of to 1674, where both a and 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, the N-terminal deletion mutations they are provided by the present invention. Such mutations include those that comprise the amino acid sequence shown in the Figure 1 (SEQ ID NO: 2) except for a deletion of at least the first 24 N-terminal residual amino acids (ie, a deletion of at least Met (1) -Glu (24)) but not more than the first 115 amino acids N-terminal residuals of Figure 1 (SEQ ID NO: 2). Alternatively, the first 24 N-terminal residual amino acids (ie, a deletion of at least Met (1) -Glu (24)) but not more than the first 103 N-terminal residual amino acids of Figure 1 (SEQ ID. NO: 2), etc., etc. In another aspect, the C-terminal deletion mutants are provided by the present invention. Such mutants include those comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) except for a deletion of at least the C-terminal residual amino acid (Ser (419)) but not more than the last 220 C-terminal residual amino acids (ie, a deletion of the residual amino acids Val (199) -Ser (419)) of Figure 1 (SEQ ID NO: 2). Alternatively, the deletion will include at least one C-terminal residual amino acid but no more than the last 216 C-terminal residual amino acids of Figure 1 (SEQ ID NO: 2). Alternatively, the deletion will include at least the last C-terminal residual amino acid but not more than the last 204 C-terminal residual amino acids of Figure 1 (SEQ ID NO: 2). Alternatively, the deletion will include at least the last C-terminal residual amino acid but not more than 192 C-terminal residual amino acids of Figure 1 (SEQ ID NO: 2). Alternatively, the deletion will include at least the last C-terminal residual amino acid but not more than the last 156 C-terminal 156 residual amino acids of Figure 1 (SEQ ID NO: 2). Alternatively, the deletion will include at least the last C-terminal residual amino acids but not more than the last 108 C-terminal residual amino acids of Figure 1 (SEQ ID NO: 2). Alternatively, the deletion will include at least the last C-terminal residual amino acids but not more than the last 52 C-terminal residual amino acids of Figure 1 (SEQ ID NO: 2). In yet another aspect, deletion mutants having amino acids deleted from both N-terminal and C-terminal residues are also included by the present invention. Such mutants include all combinations of N-terminal deletion mutants and C-terminal deletion mutants described above. The term "gene" means the segment of DNA involved in the production of a polypeptide chain; and includes the preceding and following regions of the coding region (leader and tail) as well as the intervening sequences (introns) between individual coding segments (exons). The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. It is intended that a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNAs or the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 be a fragment of ^^^^^ ^^^^ ¿Ig ^^ j ^ ^^^^ tó least about 15 nt, and more preferably at least about 200 nt way, even more preferably at least about 30 nt and so even more preferable, at least about 40 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, fragments longer than 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, 1550, 1575, 1600, 1625, 1650 or 1674 nt in length are also useful according to the present invention since they are fragments corresponding to the majority, if not all, the nucleotide sequence of the deposited cDNAs or as shown in SEQ ID NO: 1 or SEQ ID NO: 3. It is intended that a fragment of at least 20 nt in length, for example, be that which includes 20 or more contiguous bases of the nucleotide sequence of the cDNAs deposited with the nucleotide sequence as shown in SEQ ID NO: 1 or 3. In addition, representative examples of fragments of the VEGF polynucleotide 2 include, for example, fragments having a nucleotide number sequence of about 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401 -450, 451-500, 501-550, 551-600, «-AiigSá & i 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950 or 951 until the end of SEQ ID NO: 1 or the cDNA contained in the deposited clone. In this context "approximately" includes the particularly exposed, larger or smaller intervals by several nucleotides (5, 4, 3, 2 or 1), at either terminal end or both terminal ends, preferably those fragments encode a polypeptide which has biological activity The fragments of the full-length gene of the present invention can be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs, which have a high similarity of sequence with the gene or similar biological activity Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases.The probe can also be used to identify a cDNA clone corresponding to a transcript of full length and a clone or genomic clones containing the complete gene, including the regulatory and promoter regions, exons and introns. An example of a separation comprises isolating the coding region of the gene using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to separate a library of human cDNA, genomic DNA or mRNA to determine which members of the library-hybridizes the probe. A "polynucleotide" of VEGF-2 also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO: 1 or for example, cDNA clones contained in ATCC Deposits Nos, 97149 or 75698, the complement of the same. "Strict hybridization conditions" refers to an overnight incubation at 42 ° C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6) ), 5x Denhardt's solution, 10% dextran sulfate, and 20μg / ml denatured, cut salmon sperm DNA, followed by washing the filters in O.lx SSC at approximately 65 ° C. Also contemplated were nucleic acid molecules that hybridize to the VEGF-2 polynucleotides at less stringent hybridization conditions. Changes in the severity of hybridization and signal detection are mainly achieved through manipulation of the formamide concentration (lower formamide percentages result in less stringent conditions); salt conditions, or temperature. For example, less stringent conditions include an overnight incubation at 37 ° C in a solution comprising 6X SSPE (20X SSPE = 3M NaGl; NAH2P04 0.2M; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml salmon sperm DNA; followed by washes at 50 ° C with 1XXSSPE, 0.1% SDS. In addition, to achieve even less stringent conditions, the washings made after strict hybridization can be carried out at higher salt concentrations (for example 5X SSC). Note that variations in the above conditions can be achieved through the inclusion and / or replacement of alternative blocking reagents used to suppress the background in hybridization experiments. Typical blocking reagents include reagent from Denhardt, 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 that hybridizes only to polyA + sequences (such as a polyAt terminal 3 'fragments of a cDNA shown in the sequence listing), or to a complementary extension of T (c U) residues, would include the definition of the "polynucleotide", since such a polynucleotide would hybridize to any nucleic acid molecule containing a polyextension (A) or example, practically any two-stranded cDNA clone). The present application is directed to nucleic acid molecules at least 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences shown in SEQ ID NO: 1 or 3 or to the nucleic acid sequence of the deposited cDNAs, regardless of whether they code for a polypeptide having VEGF-2 activity. This is because even where a particular nucleic acid molecule does not code for a polypeptide having VEGF-2 activity, one skilled in the art would still know how to use the nucleic acid molecule, for example, as a hybridization probe or primer. of the polymerase chain reaction (PCR). 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) isolation of the VEGF-2 gene or allelic variants thereof in a library of CDNA; (2) in-situ hybridization (eg, "PEZ") to metaphase chromosomal smears to provide accurate chromosomal localization of the VEGF-2 gene, as described in Verma et al. Human Chromosomes: A Manual of Basi c Techni ques, Pergamon Press, New York (1988); and Northern Electrotransfer analysis to detect the expression of VEGF-2 mRNA in specific tissues.

Preferred, however, are nucleic acid molecules having at least 95%, 96%, 97%, 98% or 99% sequences identical to a nucleic acid sequence shown in SEQ ID NO: 1 or 3 or a sequence of nucleic acid from the deposited cDNAs which, in fact, encode a polypeptide having VEGF-2 protein activity. It is intended that "a polypeptide having VEGF-2 activity" is a polypeptide that exhibits 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: 2516-2581 (1996) and Joukov et al. , EMBO J. 5: 290-298 (1996). Of course, due to the degeneracy of the genetic code, one skilled in the art will immediately recognize that a large number of 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 cDNAs or the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 will code for a polypeptide "having VEGF-2 protein activity". In fact, since the degenerate variants of those nucleotide sequences encode all for the same polypeptide. this will be clear to any expert in the art even without j perform 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 code for a polypeptide having VEGF-2 protein activity. This is because the person skilled in the art is well aware of amino acid substitutions that are less likely or likely to not significantly affect the function of the protein (for example, the replacement of an aliphatic amino acid with a second aliphatic amino acid). For example, a guide related to how to make substitutions of phenotypically silent amino acids is provided by Bowie, J. U. et al. "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions", Science 2-7: 1306-1310 (1990), where the authors indicate that the proteins are surprisingly tolerant to amino acid substitutions. Thus, the present invention is directed to polynucleotides having at least an identity of 70%, preferably at least 90% and more preferably an identity of at least 95%, 96%, 97% or 98% with a polynucleotide that codes for the position of SEQ ID NOS: 2 or 4, as well as fragments thereof, fragments which have at least 30 bases and so "JtA-a preferable at least 50 bases and with polypeptides encoded by such polynucleotides. "Identity" per se has a recognized significance in the art and can be calculated using 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, 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).) Although there are a number of methods for measuring the identity between two polynucleotide sequences or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H., and Lipton, D., SIAM J. Applied Ma.-5: 1073 (1998)). 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 Carrillo, H., and Lipton, D., SIAM J. Applied Ma th. 48: 1013 (1998). Methods for aligning polynucleotides or polypeptides encoded in computer programs, including the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 381 (1984)), BLASTP, BLASTN, FASTA (Atschul , SF et al., J. Molec. Biol. 215: 403 (1990), Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl. 53711 (which uses the local homology algorithm of Smith and Waterman, Advances in Apllied Ma thema tics 2: 482-489 (1981)). It is intended that a polynucleotide having a nucleotide sequence at least, eg, 95% "identical "to a reference nucleotide sequence of the present invention, is the nucleotide sequence of the polynucleotide identical to the reference sequence, except that the polynucleotide sequence can include up to five mutation points per 100 nucleotides of the nucleotide sequence a reference coding for 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 total nucleotides in the reference sequence can be inserted into the reference sequence. The interrogation sequence can be a complete sequence SEQ ID NO: 1, the ORF (open reading frame), or any fragment specified as described here £ - - ------ \? ^ 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 sequence The nucleotide of the present invention can be determined conventionally using known computer programs. The preferred method for determining the best comparison between a questionable sequence (a sequence of the present invention) and an object sequence, also known as global sequence alignment), can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. . (Comp. App. Biosci. 6: 237-245 (1990)). In a sequence alignment the interrogant and objective sequences are both DNA sequences. An RNA sequence can be compared by converting the U to T. The result of such global sequence alignment is percent identity. The preferred parameters used in the FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k = tuple = 4, Mismatch Penalty = l, Union Penalty = 30, Randomization Group Length = 0, Cut Value = l, Vacuum Penalty = 5, Vacuum Size Penalty = 0.05, Window Size = 500 or the length of the object nucleotide sequence, which is always shorter. If the object sequence is shorter than the question sequence due to 5 'or 3' deletions, no M determine percent identity Deletions occur at the 5 'end of the target sequence and therefore, the FASTDB alignment does not show a similarity / 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' ends not similar / total number of bases in the questionable sequence) so that 10% of the value of the percent of identity calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final identity percent would be 90%. In another example, a 90-base object sequence is compared to a target sequence of 100 bases. This time the deletions are internal deletions, so that there are no bases in 5 'or 3' of the target sequence that are not similar / aligned with the question. In this case, the identity percent calculated by FASTDB is not corrected manually. Again, only the 5 'and 3' bases of the object sequence that are not similar / aligned with the target sequence are manually corrected. No other manual corrections are made for the purposes of the present invention. It is intended that a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a questionable amino acid sequence of the present invention, be the amino acid sequence of the "J-te-objective polypeptide identical to the interrogation sequence, except that the subject polypeptide sequence may include up to five amino acid alterations per 100 amino acids of the interrogation amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a questionable amino acid sequence, up to 5% of the residual amino acids in the target sequence can be inserted, deleted, (deleted or replaced) with other amino acids. Such alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interdispersed either individually between 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 SEQ ID NO: 2 or 4 or 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 overall comparison between a questionable sequence (a sequence of the present invention) and an objective sequence, they were not matched / aligned, as a corresponding object residue, as a percent of the total bases of the questionable sequence. If a residue has been matched / aligned it is determined by the results of the sequence alignment by the FASTDB. This percentage is then subtracted from the percent identity, calculated by the previous FASTDB program using the specified parameters, to arrive at a final identity percent value. This final identity percent value is that which is used for the purposes of the present invention. Only the N- and C-terminal residues of the target sequence, which are not the same / are aligned with the question sequence, are considered for the purposes of manual adjustment of the percent identity value. That is, only the positions of the questionable residues outside the N- and C-terminal residues furthest from the target sequence. For example, an objective sequence of 90 residual amino acids is aligned with a questionable sequence of 100 residues to determine percent identity. Deletion occurs at the N-termninal of the target sequence and therefore, the FASTDB alignment does not show a similarity / alignment of the first 10 residues at N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues in the ^^ j ^ g ^ j ^^ N- and C-terminal not similar / total number of residues in the question sequence) so that 10% of the identity percent value calculated by the FASTDB program is subtracted. If the remaining 90 residues were perfectly matched the final identity percent would be 9C! In another example, a sequence object of residue 90: s is compared with a questionable sequence of 100 residues This time the deletions are internal deletions, so there are no residues in N- or C-terminal of the target sequence that are not similar / aligned with the question. In this case, the identity percent calculated by the FASTDB is corrected manually. Again, only the positions of the residues outside the N- and C-terminal ends of the object sequence, according to what is presented by the alignment with FASTDB, that nc are similar / aligned with the interrogation sequence are manually corrected . No other manual corrections are made for the purposes of the present invention.

VEGF-2 Polypeptides The present invention also relates to polypeptides which also have the sequence! of amino acids deduced from Figures 1 or 2, having the amino acid sequence encoded by the deposited cDNAs, as well as fragments, analogues and derivatives of such polypeptides. The terms "fragment", "derivative" and "analogue", when referring to the polypeptide of Figures 1 or 2 or which is encoded by the decoded cDNA, means a polypeptide, which retains the conserved motif of the VEGF proteins as it is shown in Figure 3 and essentially the same biological function or activity. In the present invention, a "polypeptide fragment" refers to a short amino acid sequence compressed in SEQ ID NO: 2 or encoded by the cDNA contained in the deposited clone. Protein fragments can be "placed anywhere at will" or comprised within a larger polypeptide of which the fragment forms part or a region, more preferably a single contiguous region. Representative examples of polypeptide fragments of the invention include, for example, fragments with an amino acid number of about 1-20, 21-401, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280 or 281 until the end of the coding region. In addition, the polypeptide fragments may be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, or 150 amino acids in length. In this context "approximately" includes the particularly exposed, larger or smaller (5, 4, 3, 2, 1) amino acid ranges, at either end or at both ends. The polypeptide fragments include the secreted VEGF-2 proteins as well as the mature form. Additional polypeptide fragments include the secreted VEGF-2 protein or the mature form having a continuous series of deleted residues from the terminal carboxy terminal ends, or both. For example, any number of amino acids, ranging from 1 to 60, can be suppressed from the amino terminal of any secreted VEGF-2 polypeptide or mature form. Similarly, any number of amino acids, ranging from 1 to 30 can be deleted from the carboxyterminal of the secreted VEGF-2 protein or the mature form. In addition, any combination of amino and carboxy terminal deletions above are preferred. Similarly, polypeptide fragments encoding those polypeptide fragments of VEGF-2 are also preferred. Also preferred are polypeptide fragments and VEGF-2 polynucleotides characterized by structural or functional domains, such as fragments comprising an alpha helix or regions that form an alpha helix, a beta sheet region that form a beta sheet, spin regions that form a back, it spins regions that prman a spiral, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface forming regions, substrate binding regions, and high antigenic index regions. The polypeptide fragments of SEQ ID No: 2 that fall within the conserved domains are specifically contemplated by the present invention (See Figure 3). In addition, the polypeptide fragments encoding those domains are also contemplated. Other preferred fragments are biologically active fragments of VEGF-2. 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 enhanced activity, or an undesirable decreased activity. The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, synthetic polypeptides, preferably recombinant polypeptides. It will be recognized in the art that some polypeptide sequences of VEGF-2 can vary without a significant effect of the structure or function of the protein. If such differences in the sequence are contemplated, it should be remembered that there will be critical areas on the protein that will determine the activity.

In this way, the invention further includes variations of the VEGF-2 polypeptide that show substantial VEGF-2 polypeptide activity or that include regions of the VEGF protein. -2 such as the protein portions discussed below. Such mutations include deletions, insertions, inversions, repetitions and type substitutions. As indicated above, a guide related to what amino acid changes will probably be, typically silent can be found in Bowie, J.U., et al., "Deciphering the Message in Protein Sequences: Tolerance to AminoAcid Substitutions ", Science 247: 1306-1310 (1990) . Thus, the fragments, derivatives, analogs of the polypeptides of Figures 1 or 2, or those encoded by the encoded cDNAs can be: (I) one in which one or more of the residual amino acids are substituted with an amino acid residual conserved or non-conserved (preferably a residual conserved amino acid) and such substituted residual amino acid may or may not be encoded by the genetic code; or (ii) one in which one or more of the residual amino acids includes a substituent group; or (iii) one in which the mature polypeptide is fused to another compound, such as another compound to increase the half-life of the polypeptide (eg, polyethylene glycol): or (iv) one in which the amino acids Additional U * J ^ A *! * S are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for the purification of the mature polypeptide or a protein sequence; or (v) one which comprises fewer residual amino acids than those shown in SEQ ID NO: 2 or 4, and retains the conserved motif and still retains activity characteristics of the VEGF family of polypeptides. Such fragments, derivatives and analogs are considered within the scope of those skilled in the art from the teachings herein. Of particular interest are amino acid substitutions loaded with other charged amino acids and with neutrally charged neutral amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the VEGF-2 protein. The prevention of aggregation is highly desirable. The aggregation of proteins not only results in a loss of activity but is also problematic when preparing pharmaceutical formulations, because it can be immunogenic. (Pinckard et al., Clin. Exp. Immunol 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1587); Cleland et al., Cr., Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)). The replacement of amino acids can also change the selectivity of the receptor binding to the cell surface. 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 may include one or more amino acid substitutions, deletions or additions, either from natural mutations or from human manipulation. As indicated, the changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Tables 1 and 2). s The natural 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 such a polynucleotide or polypeptide could be part of a composition, and still be isolated, since such a 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, 5 0 kb, 15 kb, 10 kb or 7.5 kb in length. In one more modality, the polynucleotides of the invention comprise at least 15 continuous nucleotides of the sequence encoding VEGF-2, but do not comprise all or a portion of any intron of VEGF-2. In another embodiment, the nucleic acid comprising the sequence coding for VEGF-2 does not contain the coding sequences of a genomic flanking gene (ie, 5 'or 3' to the VEGF-2 gene in the genome). The present invention includes polypeptides of SEQ ID NOS: 2 and 4 (in particular the mature polypeptide) as well as polypeptides having a similarity of at least 70% (preferably an identity of at least 70%) with the polypeptides. of SEQ ID NOS. 2 and 4, more preferably a similarity of at least 90% (more preferably an identity of "% * 2 * minus 95%) with the polypeptides of SEQ ID NOS: 2 and 4, and even more preferably a similarity of at least 95% Even more preferably an identity of at least 905 with the polypeptides of SEQ ID NOS: 2 and 4 and also include portions of such polypeptides with such a portion of the polypeptide that generally contains at least 30 amino acids and more preferably at least 50 amino acids. As is known in the art the "similarity" between the polypeptides is determined by comparing the amino acid sequence and its conserved conserved amino acids of a polypeptide with the sequence of a second polypeptide. Fragments or portions of the polypeptides of the present invention may be employed to produce the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments can be used as intermediates to produce the full-length polypeptides. 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 mature polypeptide encoded by the deposited cDNA minus the leader (i.e., the mature protein) a polypeptide comprising approximately the amino acids . to f ----- t -23 to about 396 in SEQ ID NO: 2; a polypeptide comprising from about amino acids -22 to about 396 in SEQ ID NO: 2; a polypeptide comprising about amino acids 1 to about 396 of 1 SEQ ID NO: 2; as well as polypeptides which are at least 95% identical, and most preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above and which also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

Derivatives of VEGF-2 Natural VEGF-2 and its analogues can be further modified to contain additional chemical portions that are not normally part of the protein. Those derived portions can improve the solubility, the biological half-life or absorption of the protein. The portions can also reduce or eliminate any undesirable side effects of the proteins and the like, an overview of those portions can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. Mack Publishing Co. , Easton, PA (1990). The most suitable chemical portions for derivatization include water soluble polymers. A water soluble polymer is desirable because the protein to which it is attached does not precipitate in an aqueous environment such as a physiological environment. Preferably, the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition. A person skilled in the art will be able to select the desired polymer on the basis of such considerations as if the polymer / protoin conjugate was to be used therapeutically, and if so, the desired dose, circulation time, resistance and proteolysis and other considerations. The effectiveness of the derivation can be determined by administering the derivative, in the desired form (i.e., by means of an osmotic pump, or, more preferably, by infusion injection, or, further formulated for the oral, pulmonary, and other routes). distribution routes), and determine their effectiveness. Suitable water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / ethylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers , polyethoxylated polyols (eg, glycerol), ^^ ug &íßlM. polyvinyl alcohol, and mixtures thereof. Propylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer can be of any molecular weight, and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight ranges from about 2 kDa to about 100 kDa for ease of handling and manufacturing (the term "approximately" indicates that in polyethylene glycol preparations, some molecules will weigh more, some less, than the weight established molecular). Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of the desired sustained release, the effects, if any, on biological activity, ease of handling, the degree or absence of antigenicity, and other known effects of the polyethylene glycol on a therapeutic protein or variant). The number of polymer molecules thus bound may vary, and one skilled in the art will be able to determine the effect on the function. A mono-derivatization may be effected, or a di, tri, tetra or some combination of the derivation may be provided with the same or different chemical portions (eg, polymers, such as different polyethylene glycols weights). The ratio of polymer molecules to protein molecules (or peptide will vary, as will their concentrations in the reaction mixture). In general, the optimum ratio (in terms of efficiency or reaction in which there is no excess of unreacted protein or polymer) will be determined by factors such as the desired degree of derivation (e.g., mono, di, tri, etc.). ), the molecular weight of the selected polymer, if the polymer is branched or unbranched, and the reaction conditions. The molecules of polyethylene glycol (or other chemical portions) should be linked to the pricklyb considering the effects on the functional or antigenic domains of the protein. There are numerous joining methods available to those skilled in the art. See, for example, EP 0 401 384, the description of which is incorporated herein by reference (coupling of PEG to G-CSF), see also Malik et al. , Exp. Hema tol 20: 1028-1035 (1992) (which reports the pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently linked through residual amino acids via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule can be attached. The residual amino acids that have a free amino group can include the lysine residues and the residual N-terminal amino acids. Those that have a free carboxyl group --atfc. '. a. they may include aspartic acid residues, glutamic acid residues, and C terminal residual amino acids. Sulfhydryl groups can also be used as a reactive group to join the polyethylene glycol molecules. For therapeutic purposes, binding to an amino group, such as binding to the N-terminal or lysine group is preferred. Binding in residues important for the binding of the receptor should be avoided if binding of the receptor is desired. A chemically modified protein at the N-terminus can be specifically desired. Used in polyethylene glycol, as an illustration of the compositions herein, the ratio of polyethylene glycol molecules to protein molecules (or peptide) can be selected from a variety of polyethylene glycol molecule (by molecular weight, branching, etc.). ) in the reaction mixture, the type of pegylation reaction to be carried out, and the method for obtaining the N-terminally selected pegylated protein. The method for obtaining the N-terminally pegylated preparation (ie, the separation of this portion from other non-pegylated portions if necessary) can be by purification of the N-terminally pegylated material from a population of pegylated pro-ethylene molecules. Selective N-terminal chemical modification can be effected by reductive alkylation, which exploits the differential reductivity of different types of primary amino groups (lysine versus N-terminal) available for derivation in a particular protein. Under the appropriate reaction conditions, the substantially selective derivation of the protein at the N-terminus with a polymer containing a carbonyl group is achieved. For example, the protein can be N-terminally pegylated selectively by effecting the reaction at a pH that allows one to take advantage of the pKa differences between the epsilon-amino groups of the lysine residues and the alpha-amino group of the N-terminal residue of the protein. By such selective derivation, the binding of a water-soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of the other reactive groups occurs, such as amino groups of the side chain of lysine. Using the reductive alkylation, the water-soluble polymer may be of the type described above, and must have a single reactive aldehyde to couple to the protein. Polyethylene glycol propionaldehyde, which contains a single reactive aldehyde, can be used. The present invention contemplates the use of derivatives which are VEGF-2 expressed by prokaryotes, or variants thereof, linked to at least one molecule of polyethylene glycol, as well as the use of VEGF-2, or variants thereof, any of those known in the art of pegylation or those developed subsequently, but temperature, solvent, and pH conditions that inactivate VEGF-2 or that the variant is modified PEGylation by acylation will generally result in a protein or variant of VEGF-2 polypegilad. Preferably, the connection link will be an amide. Also, preferably, the resulting product will be substantially single (eg,> 95%) mono-, di- or tri-pegylated. However, some species with higher degrees of pegylation can be formed in amounts that depend on the specific reaction conditions used. If desired, the more purified pegylated species can be separated from the mixture, particularly the unreacted species, by standard purification techniques, including, among others, dialysis, desalination, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis Pegylation by alkylation generally involves reacting a terminal aldehyde derivative of the PEG with the protein or variant of VEGF-2 in the presence of a reducing agent. Pegylation by alkylation can also result in a protein or variant of the polypeglylated VEGF-2. In addition, the reaction conditions can be manipulated to promote pegylation substantially only at the N-terminal amino group of the VEGF-2 protein or variant (i.e., a monopegylated protein). In any case of monopegilation or polypegilation, the PEG groups are preferably linked to the protein via a -CH2-NH- group. With particular reference to the group --CH2--, this type of bond is called "alkyl" linkage here. The derivation via reductive alkylation to produce a monopegylated product exploits the different reactivity of the different types of primary amino groups (lysine versus the N-terminal) available for the derivation. The reaction is carried out at a pH which allows one to take advantage of the pKa differences between the epsilom amino groups of the lysine residues and the alpha-amino group of the N-terminal residue of the protein. By such selective derivation, the binding of a water-soluble polymer containing a reactive group such as an aldehyde, to a protein is controlled, the conjugation with the polymer takes place predominantly at the N-terminus of the protein and without significant changes occurring. of the other reactive groups, such as the amino groups of the side chain of lysine. In an important aspect, the present invention contemplates the use of a substantially homogeneous preparation of conjugated monopolymer / protein (or variant) molecules of VEGF-2 (which means that a protein or variant of VEGF-2 to which it has bound a polymer molecule substantially alone (i.e.,> 95%) in one place). More specifically, if polyethylene glycol is used, the present invention also encompasses the use of the protein or variant of the pegylated VEGF-2 which possibly lacks antigenic linking groups, and which has the polyethylene glycol molecule directly coupled to the protein or variant of VEGF-2. Thus, it was contemplated that the VEGF-2 to be used according to the present invention may include proteins or pegylated VEGF-2 variants, where the PEG groups are linked via acyl or alkyl groups. As discussed above, such products may be monopegylated or poly-pegylated (e.g., containing 2-6, and preferably 2-5, PEG groups). The PEG groups are generally linked to the protein at the alpha or epsilon amino groups of the amino acids, but it was also contemplated that the PEG groups could be attached to any amino group bound to the protein, which is sufficiently reactive to bind to a PEG group under suitable reaction conditions. The polymer molecules used in both acylation and alkylation methods can be selected from water-soluble polymers as described above. The selected polymer should be modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, "-..." fc preferably, so that the degree of polymerization can be controlled according to the provisions of the methods herein. An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is soluble in water, or C1-C10 alkoxy or aryloxy mono-derivatives (see U.S. Patent No. 5,252,714). The polymer can be branched or unbranched. For acylation reactions, the selected polymers should have a single reactive ester group. For the reductive alkylation herein, the selected polymers should have a single reactive aldehyde group. Generally, the water-soluble polymer should not be selected from natural glycosyl residues since those are usually more conveniently produced by mammalian recombinant expression systems. The polymer can be of any molecular weight, and can be branched or unbranched. A particularly preferred water soluble polymer to be used herein is polyethylene glycol. Polyethylene glycol is used herein to mean any of the PEG forms that have been used to derive other proteins, such as mono- (C1-C10) alkoxy- aryloxy-polyethylene glycol. In general, the chemical derivation can be effected under any suitable conditions used to be born to react a biologically active substance with a - "***" «« Activated polymer molecule The methods for preparing pegylated VEGF-2 variant protein will comprise the steps of (a) reacting a protein or variant of VEGF-2 with polyethylene glycol (such as a ester or reactive aldehyde derivative of PEG) under conditions whereby the protein is eluted to one or more PEG groups, and (b) obtaining the reaction products.In general, the optimal reaction conditions for the acylation reactions will be determined case by case based on known parameters and the desired result, For example, the higher the PEG: protein ratio, the greater the percentage of polypeglylated product, the reductive alkylation to produce a substantially homogeneous population of conjugated monopolymer / protein molecule ( or variant) of VEGF-2 will generally comprise the steps of: (a) reacting a protein or variant of VEGF-2 with a reactive PEG molecule under condition it is reductive alkylation, at a suitable pH that allows the selective modification of the a-amino group of the amino terminus of the protein or variant of VEGF-2; and (b) obtain the reaction products. For a substantially homogeneous population of conjugated monopolymer / protein (or variant) molecules of VEGF-2, the reaction conditions of reductive alkylation are those that allow selective binding of the water-soluble polymer portion to the N-terminus of the protein or variant of VEGF-2. Such reaction conditions generally provide pKa differences between the amino groups of the lysine and the alpha-amino group at the N-terminus (with pKa being the pH at which 50% of the amino groups are protonated and 50% not) . The pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a greater excess of polymer to protein will be desired (ie, at least reactive the N-terminal alpha-amino group, the greater the need for the polymer to achieve optimal conditions). If the pH is higher, the polymer: protein ratio does not need to be that large (ie, more reactive groups are available, so fewer polymer molecules are needed). For the purposes of the present invention, the pH will generally fall within the range of 3-9, preferably 3-6. Another important consideration is the molecular weight of the polymer. In general, the higher the molecular weight of the polymer minus the polymer molecules that can bind to the protein. Similarly, the branching of the polymer should be taken into account when optimizing these parameters. In general, the higher the molecular weight (or more the branches) the higher the polymer: protein ratio. In general, for the pegylation reactions contemplated herein, the average molecular weight is ~ - 1? R »xz * & $ from approximately 2 kDa to approximately 100 kDa. The preferred average molecular weight is from about 5 kDa to about 50 kDa, particularly preferably from about 12 kDa to about 25 kDa. The ratio of water-soluble polymer to protein or variant of VGEF-2 will generally range from 1: 1 to 100: 1, preferably (for pegylation) from 1: 1 to 20: 1 and (for monopegylation) of 1 : 1 to 5: 1.1. Using the conditions indicated above, the reductive alkylation will provide selective binding of the polymer to any copolymer or variant of VEGF-2 having an alpha-amino group at the amino terminus, (and will provide a substantially homogeneous preparation of monopolymer / protein conjugate ( or variant) of VEGF-2 The term "monopolymer / protein (or variant) conjugate of VEGF-2" as used herein means a composition comprised of a single polymer molecule bound to a protein molecule of VEGF-2 or variant protein of VEGF-2 The monopolymer / protein conjugate (or variant) of VEGF-2 will preferably have a polymer molecule located at the N-terminus, but not on the amino side groups of the lysine. of 90% monopolymer / protein conjugate (or VEGF-2 variant, and more preferably more than 95% monopolymer / protein conjugate (or variant) ) of the polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO: 2 or 3 or contained in ATCC Deposit No: 97149 or 75698 under stringent hybridization conditions or less stringent hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence described in SEQ ID NOS: 1 or 3), polynucleotide sequences of the complementary strand of a polynucleotide sequence. coding for an epitope of the invention, and polynucleotide sequences that hybridize to the complementary strand under stringent hybridization conditions or less stringent hybridization conditions defined supra. Monoclonal antibodies specific to the VEGF-2 protein have been created (SEQ ID NO: These monoclonal antibodies have been assigned the following assignments: 12E2; 13A2; 15C2; 13D6; 13E6; 19A3; 8G11; 11A8, 15E10, 9B4; 13G11 Monoclonal antibodies 15C2, 13D6 and 15E10 were deposited as a group on June 8, 1999, and were given ATCC Deposit number 198. Monoclonal antibody 13D6 was also deposited only on July 29, 1999, and He gave the ATCC Deposit Number PTA-435. The monoclonal antibodies 13A2, 13E6 and 9B4 were deposited as a group on June 8, 1999, and were given the Num. > . .., At ---- St-ik-- ATCC Deposit PTA-199. Monoclonal antibodies 8G11, 12E2 and 13G11 were deposited as a group in June 1999 and were given the ATCC Deposit Number PTA-200. The monoclonal antibodies 11A8 and 19A3 were deposited as a group on June 8, 1999, and they were given the ATCC Deposit Number PTA-201. The antibodies deposited in a mixture can be isolated on the basis of their characteristics, such as the position of the epitope map, affinity, species according to that described in the Examples. The epitopes for which the monoclonal antibodies listed above have specificity have been plotted for the VEGF-2 protein (see Figure 24). In addition, the status of each monoclonal antibody, such as the relative affinity and reactivity in ELISA and Western, 5 has been described by each of the monoclonal antibodies (see Figure 25). The term "epitope," as used herein refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and more preferably in a human. In a preferred embodiment, the present invention it encompasses a polypeptide comprising an epitope, as well as the polynucleotide which codes for this polypeptide.An "immunogenic epitope", as used herein, is defined as a portion of a protein that produces a response. - & "-" "'- -» • - * - • - • antibody in an animal, as determined by any method known in the art, eg, by methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Nati, Acad. Sci. USA 81: 3998-4002 (1983)). The term "antigenic epitope", comp is used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by immunoassays described here. Immunospecific binding excludes non-specific binding but does not necessarily exclude reactivity with other antigens. The antigenic epitopes necessarily need to be immunogenic. Fragments that function as epitopes can be produced by any conventional means (See, for example, Houghten, Proc. Nati, Acad. Sci. USA 82: 5131-5135 1985), further described in U.S. Patent No. 4, 631,211) . In the present invention, the antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, minus 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 residual amino acids in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes described herein, as well as portions thereof. Antigenic epitopes are useful, for example, to produce antibodies, including monoclonal antibodies, that bind specifically to the epitope. Preferred antigenic epitopes include the antigenic epitopes described herein, as well as any combination of two, three, four, five or more of those anti-epitope epitopes. The antigenic epitopes can be used as target molecules in immunoassays. (See, for example, Wilson et al., Cell 37: 767-778 (1984); Sutcliffe et al., Science 219: 660-666 (1983)). Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for example, Sutcliffe et al., Supra, Wilson et al., Supra, Chow et al., Proc. Nati, Acad. Sci. USA 82: 910-914, and Bittle et al., J. Gen. Virol 66: 2347-2354 (1985)). Preferred immunogenic epitopes include the immunogenic epitopes described herein, as well as any combination of two, three, four, five or more of those - * -. immunogenic epitopes. Polypeptides comprising one or more immunogenic epitopes can be presented to produce an antibody response together with a carrier prctein, such as an albumin, to an animal system (such as a rabbit or mouse) or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide can be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to produce antibodies capable of binding to, the very few, linear epitopes in a denatured polypeptide (eg, in Western electroblotting). Polypeptides containing epitopes of the present invention can be used to induce antibodies according to methods well known in the art, including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, for example, Sutcliffe et al., Supra; Wilson et al., Supra, and Bittle et al., J. Gen Virol., 66: 2347-2354 (1985). If immunization is used in vivo, the animals can be immunized with free peptide; however, the anti-peptide antibody titer can be enhanced by coupling the peptide to a macromolecular carrier, such as the lockade hemacinanin (KLH) or tetanus toxoid. For example, peptides that contain residues of . ^ and - ^^ yL cysteine can be coupled to a carrier using a binder such as the maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while the other peptides can be coupled to carriers using a more general binding agent such as glutaraldehyde . Animals such as rabbits, rats and mice are immunized with free peptides or coupled to a carrier, for example, by intraperitoneal and / or intradermal injection of emulsions containing approximately 100 μg of peptide or carrier protein and Freund's adjuvant or any other known adjuvant for stimulating an immune response. Several booster injections may be necessary, for example, at intervals of about two weeks, to provide a useful titre of antipeptide antibody that can be detected, for example, by assay by ELISA using the free peptide adsorbed to a solid surface. The anti-peptide antibody titre in serum of an immunized animal can be increased by selection of the anti-peptide antibodies, for example, by peptide adsorption on a solid support and elution of the selected antibodies according to methods selected in the art. As one of ordinary skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other 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, or any combination thereof or portions thereof). ) resulting in chimeric polypeptides. Such fusion proteins can facilitate purification or they can increase the half-life in vivo. Thus, two chimeric proteins consisting of the first two domains of the human CD4 polypeptide and several domains of the constant regions of the heavy and light chains of mammalian immunoglobulins have been shown. See, for example, EP 394,827; Traunecker et al., Nature, 331: 84-86 (19) Improved distribution of an antigen through the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding standard such such as IgG or Fc fragments (see, for example, PCT Publications WO 96/22024 and WO 99/04813). It has also been found that IgG fusion proteins having a disulfide linked dimer structure have been Disulfide bonds of the IgG portion are more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments of the same ones See, for example, Fountoulakis et al., J. Biochem., 270: 3958-3954 ( 1995) Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (eg, the brand or marker of the hemagglutinin ("HA")) to aid in the detection and expressed polypeptide urgency. For example, a system described by Janknecht et al. it allows easy purification of denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc Nati Acad Sci USA 88: 8972-197). In this system, the gene of interest is subcloned and a vaccime recombination plasmid so that the open reading frame of the gene is translationally fused to an amino terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts of the cells infected with the recombinant vaccinia virus are loaded with a column of Ni '+ nitroloacetic acid-agarose and the proteins marked with histidine can be selectively eluted with imidazole-containing buffers. Additional fusion proteins of the invention can be generated through the techniques of gene blending, motif mixing, exon mixing and / or codon mixing (collectively referred to as "DNA blending"). The mixing of the DNA can be used to modulate the activities of the polypeptides of the invention, such altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458 and Patten et al., Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, Trends Biotechnol. 16 (2); 76-82 (1998); Hansson, et al., J. Mol. Biol. 287: 265-76 (1999); and Lorenzo and Blasco, Biotechniques 24 (2): 308-13 (each of these patents and publications are incorporated herein by reference in their entirety). In one embodiment, alteration of the polypeptides corresponding to SEQ ID NO: 1 or 3 and the polypeptides encoded by those polynucleotides can be achieved by mixing the DNA. DNA mixing involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, the polynucleotides of the invention, or the encoded polypeptides, can be altered by being subjected to random mutagenesis or error prone PCR, random nucleotide insertion or other methods prior to recombination. In another modality, one or more components, motives, sections, parts, domains, fragments, etc. of a polypeptide encoding a polypeptide of the invention can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Antibodies The additional polypeptides of the invention relate to antibodies and T cell antigen receptors (TCR) which bind immunospecifically to a polypeptide, polypeptide fragment, or variant of the SEQ.

ID NO: 2 or 4 and / or an epitope of the present invention (as determined by immunoassays well known in the art to test specific antibody-antigen binding). Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, fragments produced by a library of Fab expression, anti-idiotypic (anti-ld) antibodies (including, for example, anti-ld antibodies to antibodies of the invention), and epitope binding fragments of any of the foregoing. The term "antibody", as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen-binding site that binds immunospecifically to an antigen. The immunoglobulin molecules of the present invention can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclasses of immunoglobulin molecules. More preferably, the antibodies are fragments of human antigen-binding antibodies of the present invention and include, but are not limited to, Fab, Fab 'and F (ab') 2, Fd, single-chain Fvs (scFv) , single chain antibodies, disulfide linked Fvs (sdFv) and fragments comprising any VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable regions alone or in combination with all or a portion of the following: hinge region, CH1, CH2 and CH3 domain. Also included in the invention are the antigen binding fragments which also comprise any combination of variable regions with the hinge region, the CH1, CH2 and CH3 domains. The antibodies of the invention can be of any animal origin including birds and mammals. . Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. As used herein, "human" antibodies include antibodies that have the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not about Tyr-58 to about Gly-66 in SEQ ID NO: 2; from about Gln-73 to about Glu-81 in SEQ ID NO: 2, from about Asp-100 to about Cys-108 in SEQ ID NO: 2, from about Gly-140 to about Leu-148 in SEQ ID NO: 2, from about Pro-168 to about Val-176 in SEQ ID NO: 2, from about His-183 to about Lys-191 in SEQ ID NO: 2, from about Ile-120 to about Thr-209 in SEQ ID NO: 2, from about Ala-216 to about Tyr-224 in SEQ ID NO: 2, from about Asp-244 to about His-254 in SEQ ID NO: 2, from about Gly-258 to about Glu-266 in SEQ ID NO: 2, from about Cys-272 to about Ser-280 in SEQ ID NO: 2, from about Pro-283 to about Ser-291 in SEQ ID NO: 2, from about Cys-296 to about Gln-304 in SEQ ID NO: 2, from about Ala-307 to approx. Cys-316 in SEQ ID NO: 2, from about Val-319 nasta about Cys-355 in SEQ ID NO: 2, from about Cys-339 to loop Leu-347 in SEQ ID NO: 2, from about Cys-360 to about Gl u-373 in SEQ ID NO: 2, from about Tyr-378 nasta -tf ---- a- approximately Val-386 in SEQ ID NO: 2, j of approximately Ser-388 to approximately Ser-396 in SEQ ID NO: 2, as well as polynucleotides encoding those epitopes. Antibodies that bind specifically to any epitope or polypeptide of the present invention can also be excluded. Therefore, the present invention includes antibodies that specifically bind to polypeptides of the present invention, and that allow 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 to any other analog, ortholog or homolog of a polypeptide of the present invention are antibodies that bind to polypeptides with at least 95% identity, at least 90%, at least 85%, at least 80%, at least 75%, at least 70 ° at least 65! at least 60 * at least 55 'and at least 50% (calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, the The present invention cross-reacts with murine, rat and / or homologous rabbit proteins of human and corresponding epitopes thereof. Antibodies that do not bind polypeptides with an identity of less than 95%, less > tea.*... ----- --- ----_,. '< ---- i-- of 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 % (calculated using methods known in the art and described herein) to a polypeptide of the present invention are included in the present invention. In a specific embodiment, the cross-reactivity described above is with respect to any specific antigen or unique immunogenic polypeptide, or combinations of 2, 3, 4, 5 or more of the specific antigen and / or immunogenic polypeptides described herein. Also included in the present invention are antibodies which bind to polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described to qui). The antibodies of the present invention can also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10"M, 10 -2z M, 5 X 10" JM, 10"JM, 5 X 10 M, 10 ~ 4 M, 5 X 10"5 M, 10" 5 M, 5 X 10"6 M, 10 ~ 6 M, 5 X 10 ~ 7 M, "7 M, 5 X 10" 8 M, 10"8 M, 5 X 10" 9 M, 10 ~ 9 M, 5 X 10"10 M, 10-? Or -11 M, 5 X 10" 11 M , 10_ii M, 5 X lO "1" M, 10"i¿M, 5 X 10" iJ M, 10"13 M, 5 X 10 ~ 14 M, 10 ~ 14 M, 5 X 10" 15 M or 10"15 M. The invention also provides antibodies that competitively inhibit the binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. Preferred embodiments, the antibody competitively inhibits epitope binding by at least 95, at least 90%, at least 85%, at least 80%, at menDS 75%, at-1 minus 70%, at less 65%, at least 60, at least 50% The antibodies of the present invention can act as agonists or antagonists of the polypeptides of the present invention For example, the present invention includes antibodies which perturb receptor interactions / ligand with the polypeptides of the invention either partially or totally. Preferably, the antibodies of the present invention bind to an antigenic epitope described herein, or a portion thereof. The invention presents both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent binding of the ligand but prevent activation of the receptor. Activation of the receptor (i.e., signaling) can be determined by techniques described herein or others known in the art. For example receptor activation can be determined by detecting phosphorylation (for example, tyrosine or serine / threonine) of receptor or its substrate by immunoprecipitation followed by Western electroblot analysis (for example, as described supra.). , antibodies are provided which inhibit ligand activity or receptor activity by at least 95%, by at least 90%, by at least 85%, by at least 80%, by at least 75%, by at least 70% , at least 65%, at least 60%, at least 50% of the activity in the absence of the antibody.The invention also presents receptor-specific antibodies which prevent ligand binding and receptor activation, as well as antibodies which recognize the receptor-ligand complex, and preferably, do not specifically recognize the unbound receptor or the unbound ligand.Also included in the invention are the neutralizing anti bodies which bind to the ligand and prevent binding of the ligand to the receptor, as well as antibodies that bind to the ligand, thereby preventing the activation of the receptor, but without preventing the ligand from binding to the receptor. In addition, included in the invention are the antibodies that activate the receptor. These antibodies can act as receptor agonists, that is, they potentiate or activate all or a subset of the biological activities of receptor activation mediated by the ligand, for example, by induction of receptor dimerization. The antibodies can be - & * - - * - - - '' 3 specified as agonists, antagonists or inverse agonists by biological activities comprising the specific biological activities of the peptides of the invention described herein. The above antibody agonists can be made using methods known in the art. See, for example, PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92 (6), -1981-1988 (1988); Chen et al., Cancer Res. 58 (16): 3668-3678 (1998); Harrop et al., J. Ipurunol. 161 (4) -1786-1794 (1998); Zgu et al., Cancer Res. 58 (15): 3209-3214 (1998); Yoon et al., J. Immunol. 160 (7): 3170-3179 (1998); Prat et al., J. Cell. Sci. 111 (Pt2): 237-247 (1998); Pitard et al., J. Immunol. Methods 205 (2): 177-190 (1997); Liautard et al., Cytokine 9 (4): 233-241 (1997); Carlson et al., J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman et al., Neuron 14 (4); 755-762 (1995); Muller et al., Structure 6 (9) -1153-1167 (1998); Bartunek et al., Cytokine 8 (l): 14-20 (1996) (which are hereby incorporated by reference in their entirety). The antibodies of the present invention can be used, for example, but not limited, to purify, detect and direct the polypeptides of the present invention, including diagnostic and therapeutic methods both in vi tro and in vivo. For example, antibodies have use in immunoassays to qualitatively and quantitatively measure levels of polypeptides of the present invention in biological samples. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988) (incorporated herein by reference in its entirety). As discussed in more detail below, the antibodies of the present invention may be used alone or in combination with other compositions. The antibodies can also be fused recombinantly to a N-C-terminal or chemically conjugated heterologous polypeptide (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, the antibodies of the present invention can be fused recombinantly or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins.

See, for example, PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387. The antibodies of the invention include derivatives that are modified, that is, by the covalent attachment of any type of molecule to the antibody so that the covalent binding does not prevent the antibody from generating an anti-idiotypic response. For example, but without limitation, antibody derivatives include antibodies that have been «---» - «-.« .. »'' modified, for example, by glycosylation, cetylation, pegylation, phosylation, amidation, derivation by known protecting / blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein , etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids. The antibodies of the present invention can be generated by any method known in the art. Polyclonal antibodies for an antigen of interest can Keyhole, dinitrophenol and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guer-i-n) and corinebacterium parvum. Such adjuvants are also well known in the art. Monoclonal antibodies can be prepared using a wide variety of methods known in the art including the use of hybridoma, recombinant and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988). : Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY 1981) (such references are incorporated in their entirety). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody detail in the examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such a polypeptide. Once an immune response is detected, for example, antibodies specific for the antigen are detected in the mouse serum, the spleen of the mouse is harvested and the splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells of the SP20 cell line available from the ATCC. The hibpdomas are selected and cloned by limited dilution. Hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding to a polypeptide of the invention. ascites fluids can be generated, which generally contain high levels of antibodies, immunizing mice with positive hybridoma clones. Accordingly, the present invention provides methods for generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell that secretes an antibody of the invention wherein, preferably, the hybridoma is generated by fusing the splenocytes isolated from a mouse. immunized with an antigen of the invention with myeloma cells and then separating the resulting hybridomas -.------- JM-B ---- M-5, - of the fusion of the clones of hybridomas that secrete an antibody capable of binding to a polypeptide of the invention. Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, fragments of Fab and F (ab ') 2 of the invention can be produced by proteolytic cleavage of immoglobulin molecules., using enzymes such as papain (to produce Fab fragments) or pepsin (to produce Fab (ab ') 2 fragments. F (Ab') 2 fragments contain the variable region, the constant region of the light chain and the CH1 domain of the heavy chain For example, the antibodies of the present invention can also be generated using various phage display methods known in the art In the phage display methods, the domains of the functional antibody are presented on the surface of the phage particles that contain the polynucleotide sequences encoding them In a particular embodiment, such a phage can be used to present antigen-binding domains expressed from a repertoire combined antibody library (eg, human or murine). phages expressing an antigen binding domain that bind to the antigen of interest can be selected or identified with the antigen, for example , using the labeled antigen or antigen bound or captured on a solid surface or bead. The phages used in those methods are typically filamentous phages that include the fd and M13 binding domains expressed from the phage with the Fab stabilized Fv Fv dvV domains recombinantly fused to either phage gene III or protein VII of the phage. phage Examples of phage display methods that can be used to produce the antibodies of the present invention, include those described in Brinkman et al., J. Immunol Methods 182: 41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol 24: 952-958 (1994); Persic et al., Gene 187 9-18 (1 997); Burton et al., Advances in Immunology 57; 191-280 (1994); PCT application No. PCT / GB91 / 0113; PCT publications WO 90/02809; WO 91/10737; WO 92/01407; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Patents Nos. ,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; ,780,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; ,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which are incorporated as a reference in its entirety. As described in the above references, after phage selection, the regions encoding the phage antibody can be isolated and used to generate whole antibodies, including human antibodies, or any other antigen binding fragments.

'? Yo soy. desired, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can also be employed using methods known in the art such as those described in PCT publication WO 92/22324; Mullinax et al., Biotechniques 12 (6): 864-369 (1992); and Sawai et al., AJRI 34: 24-34 (1995); and Better et al., Science 240: 1041-1043 (1988) (such references incorporated herein by reference in their entirety). Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in U.S. Patents 4,946.77 and 5,258,498; Huston et al., Methods in Enzymology 203: 46-, (1991); Shu et al., PNAS 90: 7995-7999 (1993); and Skerr¿ et al Science 240: 1038-1040 (19- For some uses, including the live use of antibodies in humans and in vi trc detection assays, it may be preferable to use chimeric, humanized or human antibodies. is a molecule in which different portions of the antibody are derived from different species of animals, such as animals having a variable region derived from a murine monoclonal antibody and a constant region of human immoglobulin.The methods for producing chimeric antisense agents are known in the art. the technique, see for example, Morrison, Science 29: 1202 1985 'Oi et al BioTechniques 4: 214 (1986); Gillies et al., (1989) J. Immunol. Methods 125: 191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are hereby incorporated by reference in their entirety. Humanized antibodies are antibody molecules of antibodies of non-human species that bind to the desired antigen having one or more regimes that determine the completeness (CDR) of the non-human species and structural regions of a molecule of human immoglobulin. Frequently, the structural residues in the human framework regions will be replaced with the corresponding residues of the CDR donor antibody to alter, preferably improvement] :, the binding of the antigen. These structural substitutions are identified by methods well known in the art, for example by modeling the interactions of the CDR and the structural residues to identify the structural residues important for the binding of the antigen and comparing the sequence to identify unusual structural residues in particular positions. (See, for example, Queen et al., U.S. Patent No. ,585,089; Riechmann et al., Nature 332: 323 (1988), which are incorporated by reference in their entirety! Antibodies can be humanized using a variety of methods known in the art including, for example, CDR grafts (EP 239,400, PCT publication WO 91/09967; US Patent Nos. 5,225,539; 5,530,101; and 5,585,089), concealment or coating (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska, et al., PNAS 91: 969-973 (1994)), and chain mixture (U.S. Patent No. 5,565,332). Fully human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be produced by a variety of methods known in the art including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also;, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated here as a reference in its entirety. Human antibodies can also be produced using transgenic mice that are capable of expressing functional endogenous immunoglobulins, but expressing human immunoglobulin genes. For example,] .si - »? - a-- human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination in mouse embryonic undifferentiated cells. Alternatively, the human variable region, the constant region, and the diversity region can be introduced into mouse embryonic undifferentiated cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be made non-functional separately or simultaneously with the introduction of human immunoglobulin sites by homologous recombination. In particular, the homozygous deletion of the JH region prevents the production of endogenous antibody. The modified embryonic non-differentiated cells are expanded and microinjected into blasts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which expresses human antibodies. The transgenic mice are immunized in a normal manner with an antigen selected for example all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized ICOS transgenic mice, using the technology of the conventional hybrid. The immunoglobulin hamana transgenes housed by the transgenic mice are rearranged during e ^ ^ ^ jig the differentiation of B cells, and subsequently undergo a class change and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology to produce hum-mos antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 825 U.S. Patent Nos. 5,413,923; 5,625,162 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318 5,885,793; 5,916,771; and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Abgenix, Inc. (Freemont;, CA) and Genpharm (San Jose, CA) can be contacted to provide human antibodies directed against a selected antigen using technology similar to that described above. Fully human antibodies that recognize a selected epitope can be generated using a technique known as "guided selection". In this method a human monoclonal antibody selected, for example, a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. (Jespers et al., Bio / technology 12: 899-903 (19) In addition, antibodies to the polypeptides of the invention can, in turn, be used to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art (See, for example, Greenspan &Bona, FASEB J. 7 (5): 437-444; (1989) and Nissinoff, J. Immunol 147 (8): 2429-2438 ( 1991).) For example, antibodies that bind to and competitively inhibit multimerization of the polypeptide and / or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the multimerization of the polypeptide and / or bind to the domain and, as a result, bind to and neutralize the polypeptide and / or its ligand. Such neutralizing anti-idio types or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize the polypeptide ligand. For example, such anticu Anti-idiotypic epos can be used to bind a polypeptide of the invention and / or to bind to its ligand-receptors, and thereby block its biological activity.Ma.

Polynucleotides Coding for Antibodies The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or less stringent hybridization conditions, for example, as defined above, to polypeptides that encode an antibody, preferably, that specifically bind to a polypeptide of the invention, so that Preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4. The polynucleotides can be obtained, and the nucleotide sequence of the polynucleotides determined, by any methods known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier et al., BioTechniques 17: 242 (1994)), which, briefly, involves the synthesis of superimposing oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplifying the bound oligonucleotides by PCR. be manipulated using methods well known in the art for the manipulation of nucleotide sequences, for example, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, for example, the techniques described in Sambrook, et al., 1990, Molecular Cloning, A Laboratoru Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, both of which are incorporated herein by reference in their entirety), to generate antibodies having a different amino acid sequence, for example to create substitutions, deletions, and / or amino acid insertions. In a specific embodiment, the amino acid sequence of the heavy and / or light chain variable domains can be inspected to identify the sequences of regions determining complementarity (CDR) by methods that are well known in the art, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs can be inserted into the framework regions, for example, into human framework regions to humanize a non-human antibody, as described supra. Structural regions may be natural or structural consensus regions, and preferably human structural regions (see, for example, Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a list of regions structural human). Preferably, the polynucleotide generated by the combination of the structural regions and the CDR codes for an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, substitutions of one or more amino acids can be made within the framework regions, and, preferably, amino acid substitutions improve the binding of the antibody to its antigen. Additionally, such methods can be used to produce substitutions or deletions of amino acids from one or more cysteine residues of the variable region that participate in an intrachain disulfide bond to generate antibody molecules lacking one or more of the intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and are within the skill of the art. In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., Proc. Nati Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature 312: 604-608 ( 1984), Takeda et al.

Nature 314: 452-454 (1985) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity As described supra, a chimeric antibody is a molecule in which they are different portions of different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, for example, humanized antibodies, alternatively, the techniques described for the production of antibodies from a single chain (U.S. Patent No. 4,946,778; Bird, Science 242: 243-42 (1988); Houston et al., Proc. Nati, Acad. Sci. USA 85: 5879-5883 (1988); and Ward et al., Nature 334: 544-54 (1989)) can be adapted to produce single-chain antibodies Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via a bone amino acid, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)).

Methods for Producing Antibodies The antibodies of the invention can be produced by any methods known in the art for the synthesis of antibodies, in particular, by chemical synthesis or, preferably, by recombinant expression techniques. The recombinant expression of an antibody of the invention, or fragment, derivative or analogue thereof, (for example, a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires the construction of an expression vector containing a polynucleotide that codes for the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain) of the invention, has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using methods well known in the art. Thus, methods for preparing a protein by expressing a polypeptide containing a nucleotide sequence encoding an antibody are described herein. Methods that are well known to those skilled in the art may be used to construct vectors of expression containing sequences coding for the antibody and appropriate transcriptional and translational control signals. These methods include, for example, recombinant DNA techniques ^ ¿^^ in vi tro, synthetic techniques and genetic recombination, in vivo. The invention thus provides reproducible vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a light or heavy chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors can include the nucleotide sequence coding for the constant region of the antibody molecule (see for example, PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody can be cloned into such vector for the expression of the entire heavy or light chain. The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells that contain a polynucleotide that encodes an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chain antibodies, the vectors encoding both heavy and light chains can be co-expressed in the host cell for the expression of a complete immunoglobulin molecule, as described below. A variety of host expression vector systems can be used to express the molecules of antibody of the invention. Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in if you These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subti l i s) transformed with bacteriophage DNA expression vectors, plasmid DNA or cosmid DNA containing sequences encoding for antibodies; yeasts (eg, Sa ccharomyces, Pi chia) transformed with recombinant yeast expression vectors containing sequences encoding antibodies; insect cell systems infected with recombinant virus expression vectors (eg, ba culoviruses) containing sequences encoding antibodies; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMjV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) ) that contains sequences that code for antibodies; or mammalian cell systems (eg, COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from human mammal (e.g., the late adenovirus promoter; 7.5K vaccinia virus promoter). More preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of the complete recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with a vector such as the promoter element of the initial major intermediate gene of a human cytomegalovirus is an effective expression system for antibodies (Foecking et al. al., Gene 45: 101 (1986), Cockett et al., Bio / Technology 8: 2 (1990)) In bacterial systems, a number of expression vectors can be advantageously selected depending on the intended use for the molecule. antibody that is being expressed. For example, when such an amount of protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the expression vector of E. coli pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)), in which the sequence encoding the antibody can be 1 ligated individually to the vector in frame with the lac Z coding region, so that it occurs a fusion protein; vectors plN (Inouye &Inouye, Nucleic Acids Res. 13: 3101-3109 (1985); Van Heeke &Schuster, J. Biol. Chem. 24: 5503-5509 (1989)); and similar. PGEX vectors can also be used to express external polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from cells used by adsorption and binding to beads of glutathione-agarose matrix followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites, so that the product of the cloned target gene can be released from the GST portion. In an insect system, the virus of the nuclear polyhedrosis Autograph californi ca (AcNPV) is used as a vector to express external viruses. The virus grows in Spodoptera frugiperda cells. Sequence _i * ^ * - j ^ íi¿? ^^ s - i - il ---------- i? í ^ ------. which encodes the antibody can be cloned individually into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter). In mammalian host, a number of virus-based expression systems can be used. In the case where an adenovirus is used as an expression vector the sequence coding for the sequence of interest is linked to an adenovirus transcription control / transduction control complex, for example, the late promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. The non-essential insertion of the viral genome (e.g., El region or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (see, eg, Logan &Shenk, Proc. Nati. Acad Sci. USA 81: 355-359 (1984)). Specific initiation signals may also be required for efficient production of the sequences coding for the inserted antibody. These signals include the ATG start eton and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can be from a variety of origins, both natural and synthetic. The efficiency of the expression can be improved by the inclusion of transcription enhancing elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol, 153: 51-544 (1987) In addition, a strain of host cells can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific manner Such modifications (eg glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein.The different host cells have specific characteristics and mechanism for post-translational processing and modification of proteins. and the genetic products.The appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the expressed external protein.To this point, the eukaryotic host cells possessing the cellular machinery for the proper processing of the primary transcript, glycosylation and Phosphorylation of the genetic product can be used. mammalian hosts include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example BT483, '-. a.Aal-. --s-i-- < ia- ---. methods are described, for example, in Ausubel et al. [eds.], Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981), which are hereby incorporated by reference in their entirety. The expression levels of an antibody molecule can be increased by amplification of the vector (for a review, see Bebbigton and Hentschel, The use of vectors based on the amplification of the gene for the expression of cloned genes in mammalian cells in the cloning of the DNA, Vol. 3 (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, the increase in the level of inhibitor present in culture of host cells will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)). The host cell can be cotransfected with two expression vectors of the invention, the first vector encoding a heavy chain derived from polypeptide and the second vector encoding a polypeptide-derived light chain. The two vectors may contain identical selectable markers that allow the equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding for and capable of expressing both heavy and light chain polypeptides can be used. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Nati Acad. Sci. USA 77: 2197 (1980)). The coding sequences for the heavy and light chains may contain cDNA or genomic DNA. Once the antibody molecule of the invention has been produced by an animal, chemically synthesized or expressed recombinantly, it can be purified by any method known in the art for the production of an immunoglobulin molecule, for example, by chromatography ( for example, affinity ion exchange chromatography, particularly by affinity for the specific antigen after protein A, and size column chromatography), centrifugation, differential solubility or by any standard technique for protein purification. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification. The present invention encompasses recombinantly or chemically conjugated antibodies (including covalent and non-covalent conjugations) with a polypeptide Proportion thereof, preferably at least 10 20, , 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily have to be directed, but it happens through linking sequences. The antibodies may be specific for antigens other than the polypeptides (or portions thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, the antibodies can be used to direct the polypeptides of the present invention to particular cell types, either in vi tro or in vi ve, by fusing or conjugating the polypeptides of the present invention to antibodies specific for surface receptors. cellular phones. Antibodies fused or conjugated to the polypeptides of the present invention can also be used in viral immunoassays and purification methods using methods known in the art.

See, e.g., Harbor et al., Supra and PCT publication WO 93/21232; WP 439.095; Naramura et al., Immunol. Lett. 3 9: 91- * * t s. 99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol. 146: 2446- 2452 (1991), which are hereby incorporated by reference in their entirety. The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention can be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to the polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain and CH3 domain or any combination of entire domains or portions thereof. The polypeptides can also be fused or conjugated to the above antibody portions to form multimers. For example, the Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonds between the Fc portions. Higher multimeric forms can be produced by fusing the polypeptides to IgA and IgM portions. Methods for fusing or conjugating the polypeptides of the present invention or portions of antibody are known in the art, see for example, U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP ^^ Ü ^ 307,434: EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Nati Acad. Sci, USA 88-.10535-10539 (1991); Zheng et al., J. Immunol. 154: 5590-5600 (1995); and Vil et al., Proc. Nati Acad. Sci, USA 89: 11337-11341 (1992) (such references are incorporated herein in their entirety). As discussed, supra, polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NOS: 2 or 4 can be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art. In addition, the polypeptides corresponding to SEQ ID NOS. 2 or 4 can be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consng of the first two domains of the human CD4 polypeptide and several domains of the heavy and light chain constant regions of mammalian immunoglobulins (EP 394,827; Traunecker et al., Nature 331: 84-86 ( 1988). Polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to IgG) may also be more efficient in binding and neutralizing other molecules, than the secreted monomeric protein or fragment thereof. protein alone, Fountoulakis et al J. Biochem 270: 3958-3964 (1995; In many cases, the Fc part in a fusion protein is beneficial for therapy and diagnosis, and can thus result, for example, in improved pharmacokinetic properties. (EP A 232,262). Alternatively, deletion of the Fc part after the fusion protein has been expressed, detected, and purified, would be desirable. For example, the Fc portion can prevent therapy or 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 Fc portions for purposes of high throughput separation assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8: 52-58 (1995); Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995) .In addition, antibodies or fragments thereof. of the present invention can be fused to marker sequences such as a peptide to facilitate purification In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the label provided in a pQE vector (QIAGEN, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available, as described in Gentz et al., Proc. Nati Acad. Sci. USA 86: 821-824 (1989), for example, hexahistidine provides convenient purification of the fusion protein. Other useful peptide tags for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)). ) and the "flag" brand. The present invention further comprises antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be diagnostic for, for example, verifying the development or progress of a tumor as part of a clinical test procedure to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substrate. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, biolumuniscentes materials, radioactive materials, positron emitting metals using various positron emission topography, and nonradioactive paramagnetic metal of ions. The detectable substance can be coupled or conjugated directly to the antibody (or fragment thereof) or indirectly, through! of an intermediate (such as, for example, a linker known in the art) using known methods n the - ^ í ÉÉÉ & vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6- mercaptopurine i, 6-thioguanine, cytarabine, 5-fluoroiracil i decarbazine), alkylating agents (eg, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine íCCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (for example, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactonopicin) (formerly actinomycin), bleomycin, mithramycin, and anthramynia (AMC)), and antimitotic agents (eg, vincristine and viblastin). The conjugates of the invention can be used to modify a given biological response, the therapeutic agent or the pharmaceutical portion should not be limited to the classical chemical therapeutic agents. For example, the pharmaceutical portion can be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, extoxina pseudomonas or diphtheria toxin, a protein such as factor of tumor necrosis, a-interferon, beta-interferon, nerve growth factor , platelet-derived growth factor, tissue plasminogen activator, an apoptotic agent, eg, TNF-alpha, TNF-beta AIM I (See, International Publication No. WO 97/33 899), AIM II (See, International Publication No. WO 97/34911). Ligand Fas (Takahashi et al., In. Immunol., 6: 1561-1574 1994)). VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, for example, angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukins, ("IL-1"), interleukin 2 ("IL-2"), interleukin 6 ("IL-6"), colony-stimulating factor of granulocytic macrophages ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors. The antibodies can also be attached to solid supports, which are particularly useful for immunoassays by purification of the target antigen. Such solid supports include, but are not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such a therapeutic moiety to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies to Drug Immunity in Cancer Therapy" in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies to Drug Distribution", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.), Pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Carriers of Antibodies to Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies? 84; Biological And Clinical Applications, Pinchera et al. (Eds.), Pp. 475-506 (1985); "Analysis, Results, and Future Prospects of the Therapeutic Use of Radioactively Marked Antibody in Cancer Therapy," in Monoclonal • Antibodies for Cancer Detection and Therapy, "Baldwin et al. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates", Im unol. Rev. 62: 119-58 (1982) Alternatively, an antibody can be conjugated to a second antibody to form a heteroconjugate antibody according to that described by Segal in US Pat. No. 4,676,980, which is incorporated herein by reference in its entirety. - & An antibody, with or without a therapeutic portion conjugated thereto, administered alone or in combination with cytotoxic factors and / or cytokines can be used as a therapeutic agent.

Immunophenotyping The antibodies of the invention can be used to immunophenotype cell lines and biological samples. The product of the translation of the gene of the present invention may be useful as a specific marker of the cell, or more specifically as a cellular marker that is differentially expressed in various stages of differentiation and / or maturation of cell types particular. Monoclonal antibodies directed against a specific epitope or combination of epitopes will allow the separation of cell populations that express the marker. Various techniques can be used using monoclonal antibodies to separate cell populations that express the markers, including magnetic separation using magnetic beads coated with antibody, "separation on a tray" with antibody bound to a solid matrix (ie, plaque), and cytometry. flow (See, for example, U.S. Patent No. 5,985,660, and Morrison et al., Cell, 96: 131-49 (1999)).

These techniques allow for the separation of particular populations of cells, such as can be found in malignant haematological processes (ie, minimal residual disease (MRD) in acute leukemic patients), and "non-self" cells in transplants to prevent graft-versus-host disease. (GVHD). Alternatively, these techniques allow the separation of undifferentiated cells and hematopoietic progenitors capable of experiencing proliferation and / or differentiation, as would be found in the blood of the human umbilical cord.

Assays for Antibody Binding The antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that may be used include but are not limited to competitive and non-competitive assay systems utilizing techniques such as western electroblots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, reactions of precipitin by gel diffusion, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name a few. Such tests are routine and are well known in 3. .? '-a ---' .fe ----.-.- ----. , -ai. ».. - -i. ' the technique (see, for example, Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, Inc., New York, which is incorporated herein by reference in its entirety). Exemplary immunoassays are briefly described below (but this is not intended as a limitation). Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as the RIPA buffer (1% NP-40 or Triton decrease the background (for example, by prepping the cell surface with sepharose beads). For further discussion regarding immunoprecipitation protocols, see, for example Ausubel et al., | Eds. , 1994, Current Protocols m Molecular Biology, Vol I, John Wiley & Sons, Inc., New York on 10.16.1. Western electro-western blot analysis generally comprises preparing prot samples, electrophoresis of the polyacrylamide gel protein samples (eg, 80-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the gel of polyacrylamide to a membrane such as nitrocellulose, PVDF or nylon, block the membrane in blocking solution (for example, PBS with 3% BSA or defatted milk), wash the membrane in wash buffer (for example, PBS-Tween 20 ), block the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, wash the membrane in wash buffer, block the membrane with a secondary antibody (which recognizes the primary antibody, for example, an anti-human antibody) conjugated to an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) or radioactive molecules (eg 32P or 1251) diluted in buffer block, wash the membrane in ~ wash buffer, and detect the presence of the antigen. One skilled in the art would recognize the parameters that can be modified to increase the detected signal and to reduce background noise. For further discussion regarding western electro-western blotting protocols, see for example Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, Vol I, John Wiley & Sons, Inc., New York on 10.8.1. ELISAs comprise preparing antigen, coating the wells of a 96-well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) to the well and incubating during a period of time, and detect the presence of the antigen. In ELISA the antibody of interest does not have to be conjugated with a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a well-detectable compound can be added. In addition, instead of coating the well with the antigen, the antibody can be coated in the well. In this case, a second antibody conjugated with a detectable compound can be added after the addition of the antigen of interest to the coated well. One skilled in the art will recognize the parameters that can be modified to increase the detected signal as well as other variations of ELISA known in the art. For one F ^^^^^ ^^^^^^^^^^^^^^^ 3 ^^^^^^^^ additional discussion regarding ELISAs, see, for example, Ausubel et al., Eds. , 1994, Current Protocols in Molecular Biology, Vol. I, John Wiley & Sons, Inc., New York, in 11.2.1. The binding affinity of an antibody to an antigen and the rate of separation of an anti-antigen-antigen interaction can be determined by competitive binding assays. An example of a competitive binding assay is a radioimmunoassay comprising the incubation of the labeled antigen (eg, 3H or 1251) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and detection of the antibody bound to the labeled antigen. . The affinity of the antibody of interest for a particular antigen in the separation rates of the binding can be determined from the data of the scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with the antibody of interest conjugated with a labeled compound (eg, 3H or 1251) in the presence of increasing amounts of a second unlabeled antibody.

Therapeutic Uses The present invention is further directed to antibody-based therapies which involve supplying antibodies of the invention to an animal, preferably a mammal, and more preferably a human patient to treat one or more of the diseases, disorders or disorders. described conditions. The therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogues and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with the expression and / or aberrant activity of a polypeptide of the invention, including, but not limited to, one or more of the diseases, disorders or conditions described here. The treatment and / or prevention of diseases, or conditions associated with the expression and / or aberrant activity of a polypeptide of the invention includes, but is not limited to, relieving symptoms associated with those diseases, disorders or conditions. The antibodies of the invention can be provided in pharmaceutically acceptable compositions as is known in the art or as described herein. The summary of the ways in which the antibodies of the present invention can be used administering antibodies, fragment derivatives, analogs or human nucleic acids to a human patient for therapy or prophylaxis. It is preferred to use high affinity and / or potency in inhibition and / or neutralizing antibodies in vivo against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments or regions will preferably have affinity for polynucleotides or polypeptides of the invention, including fragments of themselves. Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 '"M, 10" 2 M, 5 X 10 ~ 3 M, 10 ~ 3 M, 5 X 10 ~ 4 M, 10 ~ 4 M, 5 X 10"5 M, 10 ~ 5 M, 5 X 10" 6 M, 10"6 M, 5 X 10" 7 M, 10"7 M, 5 X 10 ~ 8 M, 10 ~ 8 M, 5 10 -11 -11 X 10"at M, 10" 'M, 5 X 10 ~ -1i0? M, 10"1U M, 5 X 10" 11 M, 10"X1 M, ¡5 X -12 M, 10 ~ 12 M, 5 X 10 ~ 13 M, 10 ~ 13 M, 5 X 10 ~ 14 M, 10"14 M, 5 X 10" 15 M or 10"15 M.

Genetic Therapy In a specific embodiment, nucleic acids comprising sequences coding for antibodies or functional derivatives thereof, are administered for • faU-. * --- ..- faith to treat, inhibit or prevent a disease or disorder associated with the expression and / or aberrant activity of a polypeptide of the invention, by means of gene therapy. Generic therapy refers to a therapy effected by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. For general reviews of gene therapy methods, see Goldspiel et al., Clinical Pharmacy 12: 4i 505 (1993); Wu and Wu, iotherapy 3: 87-95 (1991); Tolstcshev, Ann Rev. Pharmacol. Toxicol 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev, Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 195-215 (1993) The methods commonly used in the recombinant DNA technique that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993;; and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequences that encode an antibody, the nucleic acid sequences being pair of the expression vectors expressing the antibody or fragments of chimeric proteins or heavy or light chains thereof in a suitable guest. In particular, all nucleic acid sequences have promoters operably linked to the region encoding the antibody, such promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used which sequences encoding the antibody and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thereby providing intrachromosomal expression of the nucleic acids encoding the antibody (Koller and Smithies, Proc. Nati, Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989) .In specific embodiments, the expressed antibody molecule is a single chain antibody, alternatively, the nucleic acid sequences include sequences encoding both heavy and light chains or fragments thereof, of the antibody.

The distribution of nucleic acids in a patient can be direct, in which case the patient is exposed to the nucleic acid or vectors that include nucleic or indirect acid, in which case, the cells are first transformed with the nucleic acids in vi tro , then transplanted to the patient. These two methods are known, respectively, as genetic therapy in vivo or ex vivo.

In a specific embodiment, the nucleic acid sequences are administered directly in vivo, where they are expressed to produce the encoded product. This may be accomplished by any of numerous methods known in the art, for example, by building them as part of an appropriate nucleic acid vector and administering them so that they become intracellular, for example, by infection using defective or attenuated retroviruses or other viral vectors ( see U.S. Patent No. 4,980,286), or by direct injection of uncovered DNA, or by the use of microparticle bombardment (e.g., a genetic cannon; Biolistic,. Dupont), or by coating with lipids or cell surface receptors or I transfectants agents, encapsulation in liposomes, microparticles, or microcapsules or administering them to bind a peptide which is known to enter the nucleus, administering this one ^ yy é ^ &á to bind to an object ligand for receptor-mediated endocytosis (see, for example, Wu and Wu J. Biol Chem. 262: 4429-4432 (1987)), (which can be used to target target cell types that specifically expire receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disturb endosomes, allowing the nucleic acid to prevent lysosomal degradation. In yet another embodiment, the nucleic acid can be directed in vivo for specific adsorption and expression of the cell, by targeting a specific receptor (see, for example, PCT publications WO 93/20221, WO 92/22635, WO 92/20316; WO 93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression, by homologous recombination (Koller and Smithies, Proc Nati Acad Sci USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989)). In a specific embodiment, viral vectors containing the nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth, Enzymol, 217: 581-599 (1993)). Those Central nervous, endothelial cells and muscle Adenoviruses have the advantage of being able to infect cells that are not in division. Kozarsky and Wilson, Current Opinion in Genetics Development 3: 499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelium of the rhesus monkey. Other cases of adenovirus use in gene therapy can be found in Rosenfeld et al., Science 252: 431-434 (1991): Rosenfe Id et al., Cell 68: 143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91: 225-234 (1993); PCT Publication WO 94/12649; and Wang, et al., Gene Therapy 2: 775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

An associated adenovirus has also been proposed (AAV) for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993); U.S. Patent No. 5,436,146).

Another method of gene therapy involves transferring a gene to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection or viral infection. Usually, the transfer method includes the transfer of a selectable I marker to the cells. The cells are then placed under selection to isolate that cells that have absorbed and are expressing the transferred gene. Those cells are then delivered to a patient. In this embodiment, the nucleic acid is introduced into a cell prior to the in vivo administration of the resulting recombinant cells. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the 0 nucleic acid sequences, cell fusion, gene transfer mediated by chromosomes, genetic transfer mediated by microcells, spheroplastic fusion, etc. Numerous methods are known in the art for the introduction of external genes into cells (see, for example, Loeffler and Behr, Meth, Enzymol, 217: 599-618 (1993), Cohen et al., Meth. Enzymol. 618-644 (1993); Cline, Pharmac .. Ther 29: 69-92m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disturbed. test the stable transfer of the nucleic acid to the cells, so that the nucleic acid is expressed by the cell and , -v preferable way inheritable and expressible by its cellular progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., undifferentiated or recombinant hematopoietic cells) are preferably administered intravenously. The amount of cells contemplated for use depends on the desired effect, the condition of the patient, etc., and can be determined by those skilled in the art.

Cells into which a nucleic acid can be introduced for gene therapy purposes encompass any type of desirable available cell, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; several undifferentiated or progenitor cells, in particular undifferentiated cells or hematopoietic progenitors, for example, as obtained from the bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc, In a preferred embodiment, the cell used for gene therapy is autologous to the patient In a modality in which the recombinant cells are used in gene therapy, the nucleic acid sequences which Wwg ^^ code for an antibody are introduced into the cell, so that they are expressible by the cells or their proger.ia, and the recombinant cells are then administered ir. I live for therapeutic effect. In a specific modality, undifferentiated or progenitor cells are used. Any undifferentiated and / or progenitor cells that can be isolated and maintained in vi tro can be potently utilized according to this embodiment of the present invention (for example, see PCT Publication WO 94/08598; Stemple and Anderson, Cell 71: 973-985 (1992), Rheinwald, Meth. Cell Bio, 21A: 229 (1980), and Pittelkow and Scott, Mayo Clinic Proc. 61: 771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for gene therapy purposes comprises an inducible promoter operably linked to the r (eg coding), so that the expression of the nucleic acid is controllable by controlling the presence or inducer of the appropriate transcription.

Demonstration of the Therapeutic or Prophylactic Activity The compounds or pharmaceutical compositions of the present invention are preferably tested m vi tro and then in vivo for the desired therapeutic or prophylactic activity, before being used in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic activity of a compound or pharmaceutical composition include, the effect of a compound on a patient's cell line or tissue sample. The effect of the compound or composition on the cell line and / or tissue sample can be determined using techniques known to those skilled in the art, including, but not limited to, rosetting assays and cell lysis assays. According to the invention, in vitro assays that can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a sample of the patient's tissue is grown in culture, and is exposed to or otherwise a compound is administered, and the effect of such a compound on the tissue sample is observed.

Administration and Therapeutic Composition / Prophylactic The invention provides methods of treatment, inhibition and prophylaxis by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesirable side effects). The subject is preferably an animal, including, but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and more preferably a human.

The formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above, additional appropriate formulations and routes of administration can be selected from those described hereinafter.

Various delivery systems are known and can be used to administer a compound of the invention, for example, encapsulation of liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, for example, Wu and Wu, J. Biol. Chem. 262: 4429-4432 (1937)), construction of a nucleic acid as part of a retroviral or other vector, etc. Introduction methods include but are not limited to the intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The compounds or compositions can be administered by any convenient route, for example, by infusion or injection of a bolus, by adsorption through epithelial and mucocutaneous coatings, for example, oral mucosa, rectal and intestinal mucosa, etc. can be administered together with other biologically active agents. The administration can be systemic or local. In addition, it may be desirable to introduce the compounds or pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular or intrathecal injection.; Intraventicular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, for example, by the use of an inhaler or nebulizer, and the formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the compounds or therapeutic compositions of the invention locally to the area in need of treatment, this can be achieved, for example, and not by way of limitation, by local infusion during surgery, topical application, by example, in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, by means of an implant, the implant being a porous, non-porous or gelatinous material, including membranes such as sialastic membranes or fibers. Preferably, when a protein, including an antibody, of the invention is administered, it should be - * ^ -? * be careful in using materials which do not absjorban the protein. In another embodiment, the compound or composition can be distributed in a vesicle, in particular a liposome (see Langer, Science 249: 1527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious Diseases and Cancer, Lopez -Berestein and Fidler (eds.), Liss, New York pp. 353-365 (1989), Lopez-Berestein, ibid.m pp. 317-327, see generally ibid.).

In yet another embodiment, the compound or composition can be distributed in a controlled release system. In another embodiment, a pump may be used (see Langer, supra, Sefton, CRC Crit Ref Biomed, Emg. 14-201 1987, Buchwald et al., Surgery 507 (1980), Saudek et al., N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1 74), Controlled Bioavailability of Drugs, Design and Operation of Pharmaceutical Products, Smolen and Ball (eds.), Wiley, New York (1984), Ranger and Peppas, J., Macromol.Sci. Rev. Macromol. Chem. 23:61 (1983), see also Levy et al., Science 228: 190 (1985), During et al., Ann Neurol 25: 351 (1989), Howard et al., J. Neurosurg. - ^ jti? k & m 2m 71: 105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic agent, i.e., the brain, thus requiring only a fraction of the systemic dose (see, eg, Goodson, in Medical Contracted Release Applications, supra, vol 2, pp. 115-138 (1984)) Other controlled release systems are discussed in the Langer review (Science 249: 1527-1533 (1990)). In a specific embodiment of a compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote the expression of its encoded protein, constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, for example, by the use of a retroviral vector (see for example the No. 4,980,286), or by direct injection, or by the use of microparticle bombardment (eg, a genetic cannon; Biolistic, Dupont), or by coating with lipids or cell surface receptors or by transfecting agents, or by administration from it in a bond a peptide similar to a homeocaja known to enter the nucleus (see for example Joliot et al., Proc. Nati.

Acad. Sci. USA 88: 1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression, by homologous recombination. The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific modality the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the United States Pharmacopoeia or other pharmacopoeia generally recognized for use in animals., and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient or vehicle, in which the therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, mono-stearate * ^^ ¡^^^^ glycerol, talcum, sodium chloride, dry skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting agents or emulsifiers, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as glycerides. The oral formulation may include standard carriers such as the pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Reminton's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier to provide the appropriate form of administration to the patient. The formulation must conform to the mode of administration. In a preferred embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, the compositions , -.- .. .- «. ^ * g ^ ^^ j for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to relieve pain at the site of injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sack indicating the amount of active agent. Where the composition is to be administered by infusion, it can be distributed with an infusion bottle containing water or sterile pharmaceutical grade saline. Where the composition is administered by injection, a sterile water vial for injection or saline can be provided, so that the ingredients can be mixed before administration. The compounds of the invention can be formulated as neutral forms or salts. Pharmaceutically acceptable salts include those formed with anions such as those derived from the acids! hydrochloric, phosphoric, acetic, oxalic, tartaric, etc. , and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. -. i, ». 8 -_-- i« .-- lA ». • •""---_ twenty- The amount of the compound of the invention that will be effective in the treatment, inhibition and prevention of a disease or disorder associated with the expression and / or aberrant activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dose ranges. The precise dose to be used in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the doctor and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from model testing systems in vitro or in animals. For antibodies, the dose administered to a patient is typically 0.1 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dose administered to a patient is between 0.1 mg / kg and 20 mg / kg of the patient's body weight, more preferably from 1 mg / kg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life within the human body than antibodies of other species due to the immune response to external polypeptides. In this way, lower doses of human antibodies are often possible and a the administration of the antibodies of the invention can be reduced by increasing the absorption and penetration into the tissues (eg, in the brain) of the antibodies by modifications such as, for example, lipidation. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers may be a notice in the form prescribed by a governmental regulatory agency for the manufacture, use or sale of the drugs or biological products, notice which reflects the approval of the manufacture, use or sale for human administration by part of the agency. 1 Diagnosis and Formation of Imaging I The labeled antibodies, and derivatives and analogs thereof, that specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose or verify diseases, disorders and / or conditions associated with the expression and / or aberrant activity of a polypeptide of the invention. The invention provides the detection of aberrant expression of an individual using one or more antibodies specific for the polypeptide of interest and (b) comparing the level of gene expression with a standard level of genetic expression, thereby increasing or decreasing the level of genetic expression of the polypeptide tested compared to the level of standard expression is indicative of an aberrant expression. The invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluids of an individual using one or more antibodies specific for the polypeptide of interest and (b) comparing the level of genetic expression with a standard level of genetic expression, so that an increase or decrease in the level of genetic expression of the polypeptide assayed compared to the level of standard expression is indicative of i I a particular disorder. With respect to cancer, the presence of a relatively high amount of transcript in tissue and biopsy of an individual may indicate a predisposition I! for the development of the disease, or can provide means to detect the disease before the onset of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to use preventive measures or initial aggressive treatment. .- -. go ... 0-. --- * »-. * - t - «- n - a-4« --------- a ---. . .. TO . > * - thus preventing the development of additional cancer progress. The antibodies of the invention can be used to test protein levels in a biological sample using classical immunohistological methods known to those skilled in the art! (for example, see Jalkanen, et al., J. Cell, iBiol, 101: 976-985 (1985), Jalkanen, et al., J. Cell, Feiol, 105: 3087-3096 (1987)). Other methods based on anti-drugs useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzymatic labels, such as, glucose oxycyase; radioisotopes, such as iodine (1251, 1211); carbon (14C), sulfur (35S), tritium (3H), indium (112ln), and technesium (99Tc); luminescent marks, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. One aspect of the invention is the detection and diagnosis of a disease or disorder associated with the aberrant expression of a polypeptide of interest to an animal, preferably a mammal and more preferably a human. In one embodiment, the diagnosis comprises: a) administering (eg, parenterally, subcutaneously or intraperitoneally) to a subject an effective dose of a labeled molecule that specifically binds to the polypeptide of interest / b) wait for an interim period of time; time after administration to allow the labeled molecule to be concentrated preferentially at sites in the subject where the polypeptide is expressed (and for the unbound labeled molecule to be eliminated at a basal level); c) determine the basal level; and d) detecting the labeled molecule in the subject, such that detection of the labeled molecule above the baseline level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. The basal level can be determined by several methods including, the comparison of the amount of labeled molecule detected with a standard alue previously determined by a particular system. It will be understood that the technique that the size of the subject and the imaging system used will determine the amount of the portion subjected to the! Imaging necessary to produce diagnostic I images. In the case of a radioisotopic portion, (for a human subject, the amount of radioactivity injected will normally range from about 5 to 20 millicurieis of 99mTc. The antibody or labeled antibody fragment will then preferentially accumulate at the site of the Cells containing the specific protein The formation of images of tumors in vi Vo is described in SW Burchiel et al., "Immunopharmacokinetics of Antibodies and Their Radioactive Marked Fragments" (Chapter 13 in Tumor Imaging Formation: Radiochemical Detection of Cáíncer, S, W, Burchiel and BA Rhodes, eds., Masson Publishingj Inc.! (1982). i Depending on several variables, including the type of label used and the mode of administration, the time interval after administration to allow the labeled molecule to be concentrated preferably at sites in the subject and for the unbound labeled molecule to be eliminated at baseline is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another mode, the time interval after! of the administration is 5 to 20 days or 5 to 10 days. , In a modality, the verification of the disease or disorder is carried out repeating the method parlia the diagnosis of the disease or disease, for example,! one month after the initial diagnosis, six months later! of the initial diagnosis, one year after the initial diagnosis, etc. The presence of the labeled molecule can be detected in the patient using methods known in the in vivo venous scanning technique. Those methods depend, of! ! type of brand used. Those skilled in the art will be able to determine the appropriate method for determining a particular I-mark. The methods and devices that can be! I used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT) scan of the entire body, such as the tomography of! position emission (PET), magnetic resonance imaging (MRI), and sonography. In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a surgical instrument sensitive to radiation (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence-sensitive scanning instrument. In another modality, the molecule is marketed with a metal that emits positrons and is detected in the patient using positron emission tomography. In another embodiment, the molecule is marked with a paramagnetic mark and is detected in a patient using magnetic resonance imaging (MRI).

Equipment The present invention provides equipment that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more Ial ^ ga ^^^^^^? ^^^ gÉ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ In a specific embodiment, the compounds for the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody, which does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain means for detecting the binding of an antibody to a polypeptide of interest (eg, the antibody can be conjugated to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes that the first antibody can be conjugated to a detectable substrate). ,! In another specific embodiment of the present invention, the kit is a diagnostic kit for use in the separation of serum containing specific antibodies against proliferative and / or cancerous polynucleotides or polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit can include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one polypeptide antigen antibody. In addition, such equipment includes means for detecting the binding of the antibody to the antigen (for example, the antigen may be conjugated to a fluorescent compound such as fluorescein or rhodamine., the cul i can be detected by flow cytometry). In specific I modalities, the kit can include a polypeptide I antigen produced recombinantly or chemically synthesized. The polypeptide antigen of the equipment can also be attached to a solid support. i i In a more specific embodiment, the detection means of the equipment described above includes a solid support to which the polypeptide antigen is bound. Such equipment may also include an anti-human antibody labeled with unbound reporter. In this embodiment, the binding of the antibody to the polypeptide antigen can be detected by the binding of the antibody to the polypeptide antigen and by the binding of the labeled antibody with the reporter.; In a further embodiment, the invention includes a diagnostic kit for use in separating the I serum containing antigens from the polypeptide of the invention. ! The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means capable of detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is bound to a solid support. In a specific modality, --t-H --- the antibody can be a monoclonal antibody. The equipment detection means may include a labeled second monoclonal antibody. Alternatively, or in addition,! the detection means may include a labeled competitive antigen. In a diagnostic configuration, the test serum is reacted with a solid phase reagent having an antigen bound to the surface bound by the methods of the present invention. After binding with the antigen antibody specific for the reagent and removing the non-washed serum components, the reagent is reacted with the reporter-labeled anti-human antibody to bind the reporter to the reagent in proportion to the amount of anti-human antibody. - bound antigen on the solid support. The reagent is washed again to remove the unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically; the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometic, luminescent or colorimetric substrate (Sigma, St. Louis, MO). The reagent of the solid surface in the above test i is prepared by known techniques for joining protein material to solid support material, such as polymer beads, dip bars, 96-well plates or filter material. These binding methods generally include the non-specific adsorption of the protein to the support or the covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as a carboxyl group, hydroxyl. or activated aldehyde. Alternatively, streptavidin-coated plates can be used together! with biotinylated antigens. In this way, the invention provides a system or test equipment for carrying out this diagnostic method. The kit generally includes a support with recombinant antigens attached to the surfaces, and an anti-human antibody labeled by a reporter to detect antigen antibody bound to the surface.

Fusion Protein i Any VEGF-2 polypeptide can be used to generate fusion proteins. For example, the VEGF-2 polypeptide, when fused to a second protein, can be used as an antigenic tag. Antibodies directed 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 direct cellular locations on I base to traffic signals, the VEGF-2 polypeptides can be used as directing molecules once fused to other proteins. Examples of domains that can be fused to the VEGF-2 polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The merger does not necessarily need to be direct, but can occur through linkage sequels. In addition, the fusion proteins can also be designed to improve characteristics of the polypeptide of the VEGF-2. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the VEGF-2 polypeptide, to improve stability and persistence during purification of the host cell or subsequent handling and storage.

Also, peptide portions can be added to the VEGF-2 polypeptide to facilitate purification. Such regions can be removed before the final preparation1 of the VEGF-2 polypeptide. The addition of peptide portions to facilitate the handling of polypeptides are familiar and routine methods in the art. ! In addition, VEGF-2 polypeptides, including fragments, and epitopes specifically, can be combined with portions of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. Those proteins «--i-J-r« ---. l-t. * t fusion facilitate purification and show an increased average life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4 polypeptide and several domains of the constant region of the heavy and light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Na ture 331: 84-86 (1988)). The fusion proteins that have dimeric structures linked by disulfide (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric protein or protein fragment secreted alone. (Fountoulakis et al., J. Biochem, 270: 3958-3964 (1995)). Similarly, EP-A-O-464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising several regions of the constant portion of the immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in! A fusion protein is beneficial in therapy and diagnosis, and can thus result, for example, in improved pharmacokinetic properties. (EP-A-0332 262) Alternatively, the deletion of the Fc part after the fusion protein has been expressed, detected and purified would be desirable. For example, the Fc portion can prevent therapy and diagnosis if the fusion protein is used as an antigen for immunizations. The discovery of drugs, for example, human proteins, such as hIL-3, have been fused with Fc portions for the purpose of high-throughput separation assays to identify hIL-5 antagonists. (See, D. Bennett et al., J. Molecular Recognition 8: 52-58 (1995): K Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).

In addition, the VEGF-2 polypeptides can be fused to marker sequences, such as a peptide that facilitates the purification of VEGF-2. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (QIAQEN; Inc., 9259 Eton Avenue, Charsworth, CA, 91311), among others, many of which They are available. As described Gentz et al. , Proc. Na ti. ? cad. Scí. USA 86: 821-824 (1989), for example, hexahistidine i provides convenient purification of the fusion protein. Another useful peptide tag for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37: 767 (1984).)! Thus, any of those above mergers can be designed using the VEGF-2 polynucleotides or polypeptides. Vectors and Host Cells The present invention is also related to! recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and with host cells containing the recombinant vectors, as well as with the methods for producing such vectors and host cells and for use for the production of polypeptides or peptides of VEGF-2 by recombinant techniques.

The host cells are modified (transduolides, I transformed or transfected) genetically 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, a phage, etc. Host cells designed or transformed can be cultured in conventional nutrient media modified as appropriate to activate promoters, select 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 those skilled in the art.

The polynucleotides of the present invention can be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide sequence I can be included in any one 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; Phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccinia, I adenoviruses, poultry and pseudorrhobia virus. However, any other plasmid can be used as long as it is reproducible and viable in the host. i The appropriate DNA sequence can be inserted into the vector by a variety of methods. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site by procedures known in the art. Such procedures and others are considered within the scope of those skilled in the art. I The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence (promoter) to direct the synthesis of MRNA. As representative examples of such promoters, they are mentioned here: LTR or SV40 promoter, lac or trp of E. coli, I the PL promoter of phage lamba and other promoters that are known to control the expression of genes in eukaryotic and prokaryotic cells and their viruses. The expression vector also contains a ribosomal binding site for the initiation of I production and a transcription terminator. The vector may also include appropriate sequences to amplify the expression. ! In addition, expression vectors preferably contain at least one selectable marker gene to provide a phenotypic trait for the selection of transformed host cells. Such labels include dihydrofolate reductase (DHFR) or neomycin resistance for the culture of eukaryotic cells, and resistance to tetracycline or ampicillin for the culture of E. 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 used < 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. coli, Salmonella typhimurium, and Streptomyces; fungal cells, such as yeast; /: insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, and Bowes melanonja; and plant cells. The selection of a guest .- ...-- ^ afea- .. appropriate is considered within the scope 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 as broadly described hereinbefore. The constructs comprise a vector such as a plasmid or viral vector, in which the sequence of the invention has been inserted, in a forward or inverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Those skilled in the art know a large number of suitable vectors and promoters, and they 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, Bluescppt 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, I PSV2CAT, pOG44, pXTl and pSG available from Stratagene; and i pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to one skilled in the art. - & -. * »-.» .. »- & *, -» - * i «-.

In addition to using expression vectors in the practice of the present invention, the present invention also includes novel expression vectors comprising operator elements and promoters operably linked to nucleotide sequences that code for a protein of interest. An example of such a vector is pHE4a which is described in detail below.

As summarized in Figures 12 and 13, the components of the vector pHE4a (SEQ ID NO: 10) include: 1) a neomycin phosphotransferase gene as a selection marker, 2) a reproduction origin of E. col i, 3) a promoter sequence of phage T5, 4) two sequences of the operator c, 5) a sequence of Shine-Delgarno, 6) the repressor gene I of the lactose operon. { clq) and 7) a binding region of the multiple cloning site. The origin of reproduction (oriC) is derived from pUC19 (LTI; Gaithersburg, MD). The promoter sequence and the sequences were produced synthetically. The synthetic production of the nucleic acid sequences is well known in the art. CLONTECH Catalog 95/96, pages 215-216, CLONTECH, 1020 East Meadow Cirele, Palo Alto, CA 94303. The vector pHE4a was deposited with the ATCC on February 25, 1998, and given accession number 209645.

^ ¡¡¡* ^^^ ¿¡^ A nucleotide sequence that codes for the VEGF-2 (SEQ ID NO: 1) is operably linked to the promoter and the pHE4a operator by restricting the vector with Ndel and i any of Xbal, BamHI, Xhol, or Asp718, and isolating the largest fragment (the region of the cloning site multiple is approximately 310 nucleotides) on a gel. The nucleotide sequence that codes for VEGF-2 (SEQ ID NO: l) having the appropriate restriction sites i is generated, for example, according to the PCR protocol described in Example 1, using PCR primers having restriction sites for Ndel (as the primer). ') and either Xbal, BamHI, Xhol, or Asp718 (as the 3' primer). The PCR insert is gel purified and restricted by compatible enzymes. The insert and the vector are linked according to standard protocols.

As noted above, vector pHE4a contains a laclq gene. The laclq is an allele of the lacl gene that confers a strong regulation of the operator of the c. Amann, E. et al., Gene 65: 301-315 (1988): Stark, M., Gene 51: 255-267 (1987). The laclq gene codes for a repressor protein which binds to the lac operator sequences and blocks the transcription of the downstream (ie, 3 ') sequences. However, the product of the clq gene dissociates from the operator of the c in the presence of lactose or certain analogues of lactose for example, isopropyl B-D-thiogalactopyranoside! (IPTG). VEGF-2 is not produced in this way in this way! appreciable in non-induced host cells containing the vector pHE4a. The induction of those host cells by the! addition of an agent such as IPTG, however, results in the expression of the sequence encoding the VEGF-2. t The promoter / operator sequences of the vector pHE4a (SEQ ID NO: 10) comprise a phage T5 promoter and two operator sequences of c. One operator is located '5' to the transcription start site and the other is located 3 'to that same site. These operators, when present in combination with the product of the laclq gene, confer one! i strong repression of the downstream sequences in the absence of an inducer of the operon of the c, for example, 'IPTG.

The expression of linked sequences operatively located downstream of the lac operators can be induced by the addition of an inducer of the operon of the c, such as the IPTG. The binding of a lac inducer to the proteins of the laclq results in their release from the sequences of the operator of the c and the initiation of transcription of the sequences operably linked. The regulation of the operon of the c gene expression is checked in Devlin,! T., ^ tg ^^ tj ^ »TEXTBOOK OF BIOCHEMISTRY WlTH CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807.

The series of pHE4 vectors all contain the vector components pHE4a except the sequence encoding VEGF-2. The characteristics of vectors! pHE4a includes the optimized synthetic T5 phage promoter, the lac operator, and the Shine-Delagarno sequences.

In addition, these sequences are also optimally separated, so that the expression of an inserted gene can be strongly regulated and a high level of expression occurs after induction. ' Suitable bacterial promoters suitable for use in the introduction of proteins (of the present invention include the lacl and lacZ 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 initial CMV inp-ediate promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the retroviral LTR promoters, such as those of the Rous Sarcoma Virus (RSV), and promoters of metallothionein, such as the mouse metallothionein I promoter.

The vector pHE4a also contains the 5 'Shine-Delgarno sequence to the AUG start codon. The sequences of A & i ^ ft ^ i! ^^^^^ --- ^ - ^^^^^^^^ Shine-Delgarno are short sequences usually located at 10 nucleotides upstream (ie, 5 ') from the start codon AUG. These sequences essentially direct the prokaryotic ribosomes to the AUG start codon. In this way, ! The present invention is also directed to an expression vector useful for the production of the proteins i of the present invention. That aspect of the present invention is exemplified by the vector pHE4a (SEQ ID NO: 9).

The promoter regions can be selected from any desired gene using CAT vectors. (chloramphenicol transferase) or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particularly named bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include the immediate primary of CMV, HSV thymidine kinase, and early and late SV40, retrovirus LTR, and mouse metallothionemma I. The selection of the appropriate vector and promoter is also within the scope of those skilled in the art. In a further embodiment, the present invention relates to host cells containing the constructs described above. The host cell can be a higher 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 the bacterial cell. The introduction of the construct into the host cell can be effected by transfection with calcium phosphate, transfection mediated by DEAE-Dextran, electroporation, transduction, infection, or other methods (Davis, L., et al., Basi c Methods in Molecular ii Biology (1986)). i, The constructs in the host cells can be used in a conventional manner to produce the genetic product I encoded by the non-combining sequence. Alternatively, the polypeptides of the invention can! be produced synthetically by conventional peptide synthesizers. The mature 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 used to produce such proteins using RNA derivatives; of I DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. , Molecular Cl oning: A Labora tory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Springer Harbor, N.Y. (1989), the description of which is incorporated herein by reference. & t ^^ kÉf The transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence of the vector. Intensifiers are elements that act in the cis position of DNA, usually! i around 10 to 300 bp, which act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the reproductive originge (bp 100 to 270), an early promoter of the megalovirus site, a polyoma enhancer on the late side of the reproductive origin, and adenovirus enhancers or amplifiers .

In general, the recombinant expression vectors i will include reproducible origins and selectable markers that allow the transformation of the host cell i, for example, the ampicillin resistance gene of E. coli and the TRP1 gene of S. cerevi siae, and a promoter I derived from a highly expressed gene to direct the transcription of a structural sequence downstream. Such promoters can be derived from operons that I encode for glycolytic enzymes such as 3-phosphoglycerate kinase (PGK)., factor a, acid phosphatase, heat shock proteins, among others. The heterologous structural sequence is assembled in the appropriate phase with the translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein that includes an N-terminal identification peptide that imparts the desired characteristics, for example, stabilization or simplified purification of the expressed recombinant product. ¡I The useful expression vectors for! Bacterial use I are constructed by inserting a structural DNA i sequence coding for a desired protein together with I 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 breeding source to ensure maintenance of the vector and, if desirable, provide amplification within the host. Prokaryotic hosts suitable for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and several species within the genus Pseudomonas, Streptomyces, and Staphylococcus, although others may also be used as the material of choice. As a representative but not limiting example, the expression vectors useful for \ use . - & A-- > »,. < ------ M - "- bacterial may comprise a selectable marker or origin of bacterial reproduction derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). I Such commercial vectors include, for example, p_j-K223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMÍ (Promega, Biotec, Madison, Wl, USA). Those sections of the "skeleton" of pBR322 are combined with a suitable promoter and the structural sequence to be expressed. After transformation of a suitable host I strain and growth of the host strain to an appropriate cell density, the selected promoter can be depressed by appropriate means (e.g., temperature deviation or chemical induction) and the cells are cultured during an additional period. The cells are typically harvested by centrifugation, disturbed 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 freeze-thaw cycles, sonication, mechanical disturbance, or use of cell lysis agents.

Various mammalian cell culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 115 (1981), and other cells capable of expressing a compatible vector, eg, cell lines C127, 3T3, CHO, HeLa and BHK. The mammalian expression vectors will comprise a reproduction origin, a suitable promoter and enhancer, and also any ribosomal binding sites, polyadenylation sites, donor sites to suitable splice receptor, transcriptional termination sequences, and non-transcribed flanking sequences. 5' . DNA sequences derived from the SV40 viral genome, for example, can be used at the sites of the origin, early promoter, enhancer, splice and polyadenylation of the SV40 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, which have been modified to suppress or replace endogenous genetic material ( for example the sequence of VEGF-2), and / or to include genetic material (e.g., heterologous promoters) that are operably associated with the sequence of VEGF-2 of the invention, and that activates, alters and / or amplifies the polynucleotide Endogenous VEGF-2. For example, the method known in the art can be used to operably associate heterologous control regions and endogenous polynucleotide sequences (eg, encoding VEGF-2) via homologous recombination (see, for example, The patent United States No. 5,641,670, issued June 24, 1997; i International Application No. WO 96/29411, published on September 26, 1996; International Application No. WO 94/12650, published August 4, 1994; Koller et al. , Proc. Na ti.

Sci. USA 56: 8932-8935 (1989); and Zijlstra et al. , Na ture 342: 435-438 (1989), the descriptions of each of which are incorporated herein by reference in their entirety). The host cell may 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, the host cell may be a prokaryotic cell, such as a bacterium cell. The host strain that modulates the expression of the inserted genetic sequences, or modifies and processes the product of the gene in the desired specific form I can be chosen. The expression of certain promoters can be raised in the presence of certain inducers; in this way the expression of the genetically modified polypeptide can be controlled. In addition, the different host cells have specific characteristics and mechanisms for it! Transnational and post-translational processing and modification (for example, glycosylation, phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the expressed protein. The polypeptides can be recovered and purified from recombinant cell cultures by the methods used up to now, including precipitation with ammonium sulfate or ethanol, extraction with acid, I or anionic or cation exchange chromatography, chromatography with phosphocellulase, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. It is required to have low concentrations (approximately 0.1-5 mM) of the calcium ion present during the purification (Price et al., J.

Biol. Chem. 244: 911 (1969)). Protein refolding steps may be used, as necessary, to complete the! configuration of the mature protein. Finally, high performance liquid chromatography (pLAR) I can be used for the final purification steps. ! The polypeptides of the present invention can be a naturally purified product, or a product of chemical synthesis processes, or produced by . fi * recombinant techniques of a prokaryotic or eukaryotic host (for example, by bacteria cells, yeasts, higher plants, insects and animals in culture). Depending on the host employed in a recombinant reproduction method, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may not be glycosylated. The polypeptides of the invention may also include a residual amino acid initial methionine.

VEGF-2 Agonists and Antagonists This invention also relates to a method for selecting or separating compounds to identify those which are VEGF-2 agonists or antagonists. An example of such a method takes advantage of the capacity of the VEGF-2 to significantly stimulate the proliferation of human endothelial cells in the presence of comitogen With A. Essential 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). Con A, the polypeptides of the present invention and the compound to be selected are added. After incubation at 37 ° C, the cultures are 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 harvested on filters of fiberglass (Cambridge TechnJDlogy, Watertown, MA). The average incorporation of 3 [H] -thymidine (cpm) of cultures in triplicate using a flashing counter in liquid state (Beckman Instrurpents, Irvine, CA). The significant incorporation of 3 [H] -thymine, i compared to a control assay where the compound is excluded, indicates the stimulation of endothelial cell proliferation. To test antagonists, the assay described above was 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 a VEGF-2 antagonist. Alternatively, VEGF-2 antagonists can be detected by combining VEGF-2 and a potential antagonist with recombinant VEGF-2 receptor receptors uinitted to the membrane under conditions appropriate for a competitive binding assay. VEGF-2 can be labeled, such as by. { radioactivity, so that the number of VEGF-2 I molecules bound to the receptor can determine the effectiveness1 of the potential antagonist. \ Alternatively, the response of a second known messenger system after the interaction1 of the VEGF-2 and the receptor would be measured and compared in the presence or absence of the compound. Such second messenger systems include but are not limited to, CAMPy guanylate cyclase, ion channels or phosphoinositide hydrolysis. In another method, a mammalian cell or membrane preparation expressing the VEGF-2 receptor is incubated with labeled VEGF-2 I in the presence of the compound. The ability of the compound to intensify or block this interaction could then be measured. Potential VEGF-2 antagonists 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 that binds to the VEGF-2 receptors, however, they are inactive forms of the polypeptide and therefore prevent the action of VEGF-2. Examples of such antagonists include a negative domain mutant of the VEGF-2 polypeptide, for example, a chain of the heterodimeric form of VEGF-2 may be dominant and may mutate so that it does not retain its biological activity. An example of a negative dominant mutant includes truncated versions of a dimeric VEGF-2 which is capable of interacting with another dimer to form a Natural VEGF-2, however, the resulting homodimer is inactivated and does not exhibit the characteristic VEGF-2 activity. Another potential VEGF-2 antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control the expression of genes through the formation of a triple helix or DNA or antisense RNA, methods! both of which are based on the binding of a polynucleotide to the DNA or RNA. For example, the 5 'coding portion [of the polynucleotide sequence, which codes for | The mature polypeptides of the present invention were used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. A DNA oligonucleotide was designed that is complementary to a region of the gene involved in transcription (triple helix - see Lee et al., Nucí Acids Res. 6: 3013 (1979); Cooney e, t al, Science 241: 456 (1988) and Dervan et al., Science 251: 1360 (1991)), thus preventing the transcription and production of VEGF-2. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks the translation of the mRNA molecule into the VEGF-2 polypeptide (Antisense -Okano, J. Neurochem 56: 560 (1991); Oligodeoxynucleotides as Antisense Expression Inhibitors de Genes, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be released into cells so that the antisense RNA or DNA can be expressed to inhibit the production of VEGF-2. Potential VEGF-2 antagonists also include small molecules which do not bind to and occupy the active site of the polypeptide making Thus, the catalytic site is inaccessible to the substrate, so that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules. The antisense oligonucleotide technology i provides a novel method for the inhibition of gene expression (see generally, Agrawal I (1992) Trends in Biotech. 10: 152; Wagner (1994) Na ture 372: 333-335; and Stein et al. (1993) Science 261: 1004-1012). By mediating the binding to the complementary nucleic acid sequence (the sense strand), the antisense oligonucleotides are capable of inhibiting the splicing and translation of the RNA. In this way, the antisense oligonucleotides are capable of inhibiting the expression of proteins. Oligonucleotides have also been shown to be antisense to genomic DNA, forming a triplet, and inhibiting transcription. In addition, a sequence of murine bases 17 occurs statistically only once in the human genome, and thus the extremely precise targeting of specific sequences with such antisense oligonucleotides is possible. Antagonists can be employed 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 angiotonic activity of vascular endothelial growth factor is very useful to prevent further growth, to slow down, still! reverse the solid tumors. Although the level of expression of VEGF-2 is extremely low in normal tissues, including the breast, it may be expressed at a moderate level in at least two breast tumor cell lines 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 those disorders, antagonists can also be used to treat retinopathy associated with diabetes, rheumatoid arthritis and psoriasis. Antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described hereinabove. Truncated versions of VEGF-2 capable of interacting with natural VEGF-2 can also be produced to form dimers to cause the failure of cell growth activation. endothelial, thereby inactivating endogenous VEGF-2. 0, mutant forms of VEGF-2 that form dimers with each other and occupy the ligand binding domain of the appropriate tyrosine kinase receptors on the surface of the target cell, but do not activate the cell growth. Alternatively, antagonists may be employed for the polypeptides of the present invention that bind to the receptors to which a polypeptide of the present invention binds normally. The antagonists can be closely related proteins, so that they recognize and bind to the natural prointein receptor sites, however, they are inactive forms of the natural protein and therefore prevent the action of VEGF-2 since the receiving sites are busy. In this way, the action of VEGF-2 is prevented and the antagonists / inhibitors can be used therapeutically as a drug! antitumor occupying receptor sites of tumors that are recognized by VEGF-2 or by inactivating VEGF-2 itself.

Antagonists / inhibitors can also be used to prevent inflammation due to the action of increasing vascular permeability VEGF-2. Antagonists / inhibitors can also be used to treat the growth of solid tumors, diabetic retinopathy, psoriasis, and rheumatoid arthritis. i! Antagonists / inhibitors can be employed in a composition with a pharmaceutically acceptable carrier, for example, as described herein above. In addition, as shown in Example 9, i-specific antibodies to VEGF-2 can be combined with VEGF-2 polypeptides to increase the response of endothelial cells. Endothelial cells that respond to a combination of VEGF-2 polypeptide and VEGF-2 specific antibodies include vascular or lymphatic vessels. The combination of VEGF-2 specific antibodies and VEGF-2 polypeptide can be used! to treat individuals who need an increase in the proliferation of endothelial cells, angiogenesis and / or lymphangiogenesis, as described by the specification.

I Therapeutic Applications of VEGF-2 i As used in the following section ^ "VEGF-2" is intended to refer to the full length and mature forms of the polynucleotides and polypeptides of the. VEGF-2 described herein and to the analogous polynucleotides and polypeptides, derivatives and mutants of VEGF-2 described herein.

G ^ & ? The VEGF-2 polypeptide of the present invention is a mitogen for photoreceptor cells. As shown in Figures 12-15, VEGF-2 increases the number of cells, cell survival, rhodopsin expression, and the number of rhodopsin cells in retinal cultures. It can be used to treat eye disorders, including damage and diseases, including angioid events, retinitis pigmentosa, Kearn syndrome, pigmented pattern dystrophies, retinal perforations, retinitis, chorioretinitis, cytomegalovirus retinitis, acute retinal necrosis syndrome. , central alveolar choroidal dystrophy, dominant drusen, hereditary hemorrhagic macular dystrophy, macular dystrophy of North Carolina, pericentral cdroidal dystrophy, adult foveomacular dystrophy, benign concentric ring macular dystrophy, central aureolar epithelial dystrophy, congenital macular coloboma, macular edema I dominant hereditary quistoid, retinoeschistosis foveal family, distr bright fenestrated macular devia, progressive foveal dystrophy, slowly progressive macular dystrophy, pseudoinflammatory Sorsby dystrophy, cones-rod dystrophy, progressive cone dystrophy, congenital Leber amaurosis, Goldman-Favre syndrome, Bardet-Biedl syndrome , Bassen-Kornzweig syndrome and Bassen-Kornzweig (abetalipoproteinemia), disease of! Best (viteliform dystrophy), choroidemia, spin atrophy, congenital amaurosis, Refsum syndrome, Stargardt's disease and Usher syndrome. Other retinopathies that may benefit from the administration of VEGF-2 include age-related macular degeneration (in its dry and wet forms), diabetic retinopathy, peripheral vitreoretinopathies, photonic retinopathies, surgery-induced retinopathies, viral retinopathies (such as HIV-related retinopathy for AIDS), ischemic retinopathy, retinal detachment and traumatic retinopathy. VEGF-2 can be administered together with other proteins which sor therapeutics for eye cells, including, but not limited to: retic acid, mitogens such as insulin, insulin-like growth factors, growth factor epidermal, vasoactive growth factor, adenylate cyclase activating polypeptide of the pituitary and somatostátina; neurotrophic factors such as the neurotrophic factor derived from the glial cell line, brain-derived neurotrophic factor, neutrophil-3, neutrophin-6, insulin-like growth factor, ciliary neurotrophic factor, growth factors and fibroblastic acids and basic, fibroblastic growth factor 5, transformation growth factor > «* £» »- beta, and transcripts regulated by cocaine-amphetamine (CART); and other growth factors such as epidermal growth factor, leukemia inhibitory factor, interleukins, interferons and colony stimulating factors; as well as molecules and materials that are functional equivalents of those factors. Additionally, antibodies can also be used in an immunoassay to detect the presence of tumors in certain individuals. Enzyme immunoassays can be made from blood samples of an individual. i Elevated levels of VEGF-2 can be considered cancer diagnosis.

Pharmaceutical Compositions i VEGF-2 polypeptides and polynucleotides of the present invention can be used in combination with an acceptable pharmaceutical carrier to comprise a pharmaceutical composition. Such compositions comprise a therapeutically effective amount of the polypeptide,! I polynucleotide, agonist or antagonist and a pharmaceutically acceptable carrier or receptor. Such carriers include, but are not limited to, antioxidants, preservatives, dyes, flavorings and diluents, emulsifying agents, suspending agents, solvents, fillers, and additives, buffers, release vehicles, diluents, excipients and / or adjuvants. pharmacists The formulation must conform to the mode of administration. For example, suitable carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The primary solvent in a vehicle may be aqueous or non-aqueous in nature, and the vehicle may contain other pharmaceutically acceptable excipients to modify or maintain pH, osmolarity, viscosity, clarity, color, sterility, stability, speed. of dissolution or odor of the formulation Similarly, the vehicle can contain other pharmaceutically acceptable excipients to modify or maintain the velocidaid release of VEGF-2, or to promote the absorption or penetration of VEGF-2 through the membranes of the Such excipients are those substances commonly and commonly used to formulate doses for parenteral administration in their unit dose or multiple dose form.After the therapeutic composition has been formulated, it can be stored in sterile bottles with a solution. , suspension, gel, emulsion, solid or powder i dehydrated or lyophilized. r stored in a ready-to-use form or in a form, for example, lyophilized, which required reconstitution before administration.

^^ | The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions I of the invention. Associated with such containers, a notice may be found in the form prescribed by a governmental regulatory agency for the manufacture, use or sale of pharmaceutical or biological products, notice which reflects approval for the manufacture, use or sale for human administration by the agency. In addition, the I polypeptides, agonists and antagonists of the present invention can be used in conjunction with other therapeutic compounds. The VEGF-2 polypeptide or polynucleotide can be administered in pharmaceutical compositions j and in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily use of the pharmaceutical compositions of the present invention will be decided by the attending physician within the scope of prudent medical judgment. The specific therapeutically effective dose level i for any particular patient will depend on a variety of factors including the type and degree of response to be achieved; the specific composition and other agents, if any, employed, the age, body weight, general health, sex and diet of the patient;I the time of administration, route of administration,! and the rate of excretion of the composition; the duration of the treatment; drugs (such as the chemotherapeutic agent) used in combination or in a manner coincident with the specific composition; and similar factors well known in the medical arts. Suitable formulations, known in the art, can be found in Remington's Pharma ceuti cal Sciences (latest! Edition), Mack Publishing Company, Easton, PA. i The composition of VEGF-2 to be used in therapy would be formulated and dosed in a manner consistent with a! good medical practice, taking into consideration the clinical condition of the individual patient (especially the side-effects of VEGF-2 treatment only), the patient's release of the VEGF-2 composition, the method of administration, the scheduling of the administration, and ii other factors known to physicians. The "effective amount" of VEGF-2 for the purposes herein is determined in this way by such considerations. ! The pharmaceutical compositions may be administered in a convenient form, such as by the oral, topical, intravenous, intraperitoneal, intramuscular, intraarticular, subcutaneous, intranasal, intratracheal, intraocular or intradermal routes. The compositions effective to treat and / or for the prophylaxis of the specific indication. In most cases, the dose of VEGF-2 is about 1 μg / kg to about 30 mg / kg of body weight daily, taking into consideration the routes of administration, symptoms, etc. However, the 'dose It can be as low as 0.001 μg / kg. For example, in the specific case of topical administration the doses are preferably administered from about 0.01 μg to 9 mg per cm2. In the case of intraocular administration, 1 the doses are preferably administered, about 0.001 μg / ml to about 10 mg / ml, and more preferably from about 0.05 mg / ml to about 4 mg / ml. As a general proposal, the total pharmaceutically effective amount of VEGF-2 administered parenterally by more preferable dose will be in the I range from about 1 μg / kg / day to 100 mg / kg / day of patient body weight, although, As noted above, this will be subject to therapeutic discretion. If given continuously, VEGF-2 is typically administered at a dose rate of about 1 μg / kg / hour to about 50 μg / kg / hour, either for 1-4 injections per day or continuous subcutaneous infusion, for example, using a minibmba. -sfc ¡.Al-i Aí¡s¿y ^ H & A bagged solution or intravenous bottled solution may also be used. VEGF-2 is also administered in an appropriate manner by sustained release systems. Examples of sustained release compositions include semipermeable polymer matrices in the form of shaped articles, eg, films or microcapsules. Sustained release matrices include polylactides (U.S. Patent No. 3,773,919, EP 58,881), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers 22: 541-556 (1983 )), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed, Mater X Res. 15: 161-211 (1981), and R. Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) Or poly-D- (-) -3-hydroxybutyric acid (EP 133,988). Sustained-release VEGF-2 compositions also include VEGF-2 entrapped in liposomes. Liposomes containing VEGF-2 are prepared by methods known per se: DE 3,218,121; Epstein, et al., Proc. Nati Acad. USA 82: 3688-3692 (1985); Hwang et al., Proc. Nati Acad. Sc EUA t 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Patent Application 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; And EP 102,324. Commonly, the liposomes are of a type ^^^ small unilamellar (approximately 200-800 Angstroms) in which the lipid content is greater than approximately mol percent of cholesterol, with the selected proportion adjusted for optimal VEGF-2 therapy. For parenteral administration, in one embodiment, VEGF-2 is formulated in a general manner by mixing it into the desired degree of purity, in an injectable unit dosage form (solution, suspension or emulsion), with a pharmaceutically acceptable carrier, is say, one that is not toxic to the receptors at the doses and concentrations used and that is compatible with other ingredients of the formulation. For example, formulation I preferably does not include oxidizing agents and! other compounds known to be harmful to the polypeptides. In certain embodiments, VEGF-2 is administered orally. VEGF-2 that is administered in this way > it can be encapsulated and can be formulated with or without those carriers commonly used in the composition of solid dosage forms. The capsule can be designed to release the active portion of the formulation at the point 'in the gastrointestinal tract when the bioavailability is maximized and the presystemic degradation is minimized.

Additional excipients may be included to facilitate the uptake of VEGF-2. Diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, agents can also be used. biocompatible, granules or ported in contact lenses. The intraocular composition may also contain a physiologically compatible ophthalmic vehicle that those skilled in the art may select using conventional criteria. The vehicles can be selected from known ophthalmic vehicles which include but are not limited to water, polyethers such as polyethylene glycol 400, polyvinyls such as polyvinyl alcohol, povidone, cellulose derivatives such as carboxymethylcellulose, methylcellulose and hydroxypropyl methylcellulose, petroleum derivatives such as mineral and white petrolatum, animal fats lanolin, vegetable fats such as peanut oil, acrylic acid polymers such as carboxymethyl glycol, polysaccharides such as dextrans and glycosaminoglycans such as sodium and potassium chloride, chloride, zinc chloride and buffers such as sodium bicarbonate or sodium lactate. I can also use high molecular weight molecules. The physiologically compatible condoms that do not inactivate VEGF-2 present in the composition include alcohols such as chlorobutanol, benzalkonium chloride and EDTA, or any other suitable preservative known to those skilled in the art. For example, VEGF-2 can be administered intraocularly directly from about 1 mg / eye to about 1 mg / eye in a single injection or multiple injections. The formulation of topical ophthalmic preparations, including solutions, suspension and ophthalmic ointments are well known to those skilled in the art (see Remington's Pharmaceutical Sciences, 18th Edition, Chapter 86, pages 1581-1592, Mack Publijshing I Company, 1990). Other modes of administration are available, including intracameral injections (which can be done directly in the anterior chamber or directly in the vitreous chamber), injections! subconjunctival and retrobulbar injections, and the methods and means for producing suitable ophthalmic preparations for such modes of administration are well known. VEGF-2 1 can also be administered in the subretinal space between the photoreceptor layer and the pigmentary and retinal epithelial layers. I As used here, "extraocular" refers to the ocular surface and the (outer) space between the globd of the eye and the eyelid. Examples of extracellular regions include the fornix or cul-de-sac of the eyelid, the conjunctival surface and the corneal surface. This location is external to all eye tissue and an invasive procedure is not required to access this region. Examples of extraocular systems include inserts and drops, gels or I ointments applied "topically", which can be used to release the therapeutic material to those regions. The extraocular devices are generally easily removable, even by the patient. [The following patents describe extraocular systems which are used to administer drugs to the extracellular regions. Higuchi et al. discloses in U.S. Patent Nos. 3,981,303, 3,986,510 and 3,995,635 a biodegradable eye insert which contains a drug. The insert can be made in different ways to be retained in the cul-de-sac of the eyeball, the extraocular space between the eyeball and the eyelid. Various common biocompatible polymers are described as suitable for use in the manufacture of this device. These polymers include (zinc, poly (lactic acid) alginate, poly (vinyl alcohol), poly (anhydrides) and poly (glycolic acid) .The patents also describe I membranes coated devices with permeation. i ^^^ ife ^^^ | ^^^^^? "& gj¡ ^^ # ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Those spos t vps are placed extraocularly in the ocular cul-de-sac. Among the polymeric systems of interest are poly (vinyl chloride) -co-poly (vinyl acetate) copolymers. Kaufman discloses in U.S. Patent Nos. 4,865,846 and 4,882,150 an ophthalmic drug delivery system containing at least one bioadjuvant ointment portaadpr material for the conjunctival sac. The patent describes polymeric systems, such as poly (lactide), poly (glycolide), poly (vinyl alcohol) and cross-linked collagen with suitable release systems. In the presently described use of VEGF-2 for the treatment of diseases or retinal damage it is also advantageous that the topically applied ophthalmic formulation includes an agent to promote the penetration or transport of the therapeutic agent in the eye. Such agents are known in the art. For example, Ke et al. , US Patent No. 5,221,696 describes the use of materials to improve the penetration of ophthalmic preparations through the cornea.

Infraocular systems are those systems that are suitable for use in any tissue compartment within, between or around the tissue layers of the eye itself. These sites include the subcojunctival (under the ocular mucosa membrane adjacent to the globe of the eye), orbital (behind the eyeball) and intracameral (inside the balloon chambers of the o itself). In contrast to the extraocular systems, an invasive procedure consists of injection or implantation. it is required to have access to those regions. i I The following patents describe devices! infraocular Wong, US Patent No. 4,853,224, I describes microencapsulated drugs to be introduced | in the eye chamber. The polymers that are used in this system include polyesters and polyethers. Lee, US Patent No. 4,863,457, describes a biodegradable device which is surgically implanted intraocularly for the sustained release of therapeutic agents. The device is designed to patch surgical implantation under the conjunctiva (mucous membrane t of the eyeball). Krezancaki, U.S. Patent No. 4,188,373, discloses a pharmaceutical vehicle which gellifies the temperature of the human body. This vehicle is an aqueous suspension of the drug and synthetic gums or derivatives US Nos. 4,474,751 and 4,474,752 a polymer-drug system which is liquid at room temperature and gels at body temperature. Suitable polymers used in this system include polyoxyethylene and polyoxypropylene. Davis et al. discloses in US Pat. No. 5,384,333 a biodegradable, injectable drug delivery polymer which provides long-term drug release. The drug composition is comprised of a pharmaceutically active agent in a biodegradable polymer matrix, wherein the polymer matrix is a solid at temperatures in the range of 20EC to 37EC, and flows at temperatures in the range of 38EC to 52EC. The polymer that releases drug no I is limited to the release of liquid-soluble drug formulations. For example, the polymer can be used as a matrix to stabilize and retain at the injection site microspheres, liposomes or other drugs attached to particles at the site of injection. A particularly suitable vehicle for intraocular injection is sterile distilled water in which VEGF-2 is formulated as an isotonic, sterile, appropriately preserved solution. Another ophthalmic preparation plus i may involve the formulation of VEGF-2 as an agent such as injectable microspheres or liposomes, which provide for the slow or sustained release of protein that can Then, they are released as an injection reservoir. Other suitable methods for the intraocular introduction of VEGF-2 include implantable drug delivery devices containing VEGF-2. The ophthalmic preparations of the present invention, particularly the topical preparations, may include other components, for example ophthalmologically acceptable preservatives, toning agents,! cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, antioxidants and surfactants, as is well known in the art. For example, suitable tonicity improving agents include alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol and the like. Advantageously, sufficient tonicity enhancing agent is added, so that the formulation to be instilled into the eye is hypotonic or substantially isotonic. Suitable condoms include, but are not limited to, benzalcpnio chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, and the like. I can also be used hydrogen peroxide as a preservative. The beta-cyclodextrin. Suitable surfactants or wetting agents include, but are not limited to, sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol, and the like. The dampers can be conventional dampers I such as borate, citrate, phosphate, bicarbonate or tris-HCl. The components of the formulation are present in concentrations that are acceptable for the site of extraocular or infraocular administration. For example, shock absorbers are used to maintain the composition! at physiological pH I or at a slightly lower pH, typically within a pH range of about 5 to about 8. Additional formulation components may include materials which provide prolonged ocular residence of the extraocularly administered therapeutic agent to maximize the Topical contact and promote absorption. Suitable materials include polymers or I I I gels that form materials which provide greater viscosity to the ophthalmic preparation. Chitosan is a particularly suitable material as an agent for! I control the rate of ocular release in formulations I of sustained release liquid ophthalmic drugs (see U.S. Patent No. 5,422,116, Yen, et al.). The suitability of the formulations of the present invention for controlled release (e.g., sustained and prolonged release) of an ophthalmic treatment agent in the eye can be determined by various methods known in the art, e.g., as described in Journal of Con trolled Relay se 6: 361-313, 1987, as well as variations thereof. Another, more ophthalmic preparation may involve an effective amount of VEGF-2 in a mixture with non-toxic ophthalmically acceptable excipients which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or other suitable vehicle, ophthalmic solutions can be prepared in unit dosage form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binding agents, such as starch, gelatin or acacia, lubricating agents such as magnesium stearate, stearic acid or talc. In general, the formulations are prjeparan putting in contact the VEGF-2 evenly and intimately with liquid carriers or finely divided solid carriers i or both. Then, if necessary, the product will form in the desired formulation. Preferably the carrier is a parenteral carrier, more preferably, a solution that is isotonic with the recipient's blood. Examples of such carriers include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles such as distilled oils and! Ethyl oleate are also useful here, as well as liposomes. Suitable formulations, known in the art, can be found in Remmgton's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, PA. The carrier suitably contains minor amounts of additives such as substances that increase isotonicity and chemical stability. Such materials are not toxic to the receptors and the doses and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts; antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues, for example, polyarginine or tripeptides; proteins, such as seroalbumin, gel tub, I or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid or arganim, monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins, chelating agents I such as EDTA, sugar alcohols such as mannitol or sorbitol, counterions such as sodium, and / or nonionic surfactants such as polysorbates, poloxamers or PEG. VEGF-2 is typically formulated in such vehicles at a concentration of approximately 0.01 μg / ml at 100 mg / ml, preferably from 0.01 μg / ml to 100 mg / ml, at a pH of about 3 to 8. It will be understood that the use of | certain of the excipients, carriers or stabilizers i above will result in the formation of salts of the VEGF-2. I VEGF-2 to be used for therapeutic administration must be sterile. Sterility is easily achieved by filtration through sterile filtration membranes (eg, 0.2 micron membranes). (The VEGF-2 therapeutic compositions are generally I placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a plug pierceable by a hypodermic injection needle. 2 will commonly be stored in unit or multi-dose containers, for example, sealed vials or flasks, as an aqueous solution or as a freeze-dried formulation for reconstitution.As an example of a lyophilized formulation, 10 ml bottles are i filled with 5 ml of filtered VEGF-2 aqueous solution, ^^ ¡1 ^^ sterile at 1% (weight / volume), and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized VEGF-2 using water for! bacteriostatic injection.

Genetic Therapy Methods Another aspect of the present invention is the methods of gene therapy to treat disorders, diseases and conditions. Genetic therapy methods are related to the introduction of nucleic acid sequences (DNA, RNA and antisense DNA or RNA) in an animal to achieve expression of the VEGF-2 polypeptide of the present invention. This method requires a polynucleotide, which codes for a VEGF-2 polypeptide operably linked to a promoter and any other genetic elements necessary for expression of the polypeptide for the target tissue. Such genetic therapy and methods of distribution or release are known in the art, see for example, WO90 / 11092, W098 / 11779; U.S. Patent Nos. 5693622,5705151, 5580859; Tabata f} -. et al. (1997) Cardiovasc. Res. 35 (3): 470-479, Chao, J. et al. i (1997) Pharma col. Res. 35 (6): 511-522, Wolff, J.A. (1997) Neuromuscul. Disord. 7 (5): 314-318, Schwartz, B. et al. (nineteen ninety six) Gene Ther. 3 (5): 405-411, TsuTumi, Y, et al., 1996) Circulation 94 (12): 3281 -3290 (incorporated herein by reference).

As discussed more fully below, the polynucleotide sequences of VEGF-2 preferably have a therapeutic effect after being absorbed by a cell. Examples of polynucleotides that are themselves therapeutic are antisense DNA and RNA; the DNA encoding an antisense RNA; or the DNA that codes for the tRNA or rRNA to replace deficient endogenous molecules. For example, a promoter can be operably linked to an ed AD sequence encoding an antisense RNA. The antisense RNA oligonucleotide hybridizes to the mRNA in vi vo of a mRNA molecule in a polypeptide (Okano, J. Neurochem 56: 560 (1991)). The antisense RNA must be of full length and complementary to prevent the translation of its target mRNA. | Thus, for example, the cells of a patient can be modified with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of VEGF-2 ex vivo, with the modified cells being then provided to a patient to be treated with the polypeptide. Such methods are well known in the art. See for example Belldegrun, A1., Et 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 that are modified are photoreceptor cells. These modified cells can be reintroduced into the patient through injection to the tissue of origin, the tissues that roll the tissue of origin, veins or arteries, or through catheter injection. In one embodiment, the modified cells are attached to the sclera to produce and release protein from the I! VEGF-2 directly in the vitreous humor.

Transplant studies of photoreceptor cells designed to replace defective or lost cells due to diseases or retinal damage in animal models of retinal degeneration have been successfully carried out (Silverman and Hughes, Invest, Ophthalmol, Vis.Sci.30: 1684-1690). (1989), Gouras et al., Neuro-Ophthalmol 10: 1 65-1 76 (1990)). It was contemplated that photoreceptor I cells can be obtained from donor eyes and ** ^ ^ Keep in cultivation as described here. The cells would then be used as a source of purified photoreceptors to be transplanted via the subretinal space in the retina of patients suffering from retinal damage or disease. These patients will be treated with immunosuppressive tetapias I to eliminate immune responses and rejection of the grafted cells. The retinas of the ex vivo donor will be cultivated in the presence of VEGF-2 to improve its growth and survival. Patients who will receive photoreceptor cell transplants will be treated with the Intravitreal VEGF-2 necessary to promote the survival and maturation of grafted photoreceptors. As discussed in more detail below, the polynucleotide constructs of VEGF-2 can | be distributed by any method that distributes materials and injectables to the cells of an animal such as, injection in the interstitial space of tissues (heart, muscle, skin, lung, liver and the like). The polynucleotide constructs of VEGF-2 can be distributed in a pharmaceutically acceptable liquid. In one embodiment, the polynucleotide of VEGF-2 is I distributed as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to I i sequences that are free of any delivery vehicle that acts to aid, promote or facilitate entry into the cell, including viral sequences, viral particles, formulations liposomal, lipofectin or precipitating agents and the like. However, VEGF-2 polynucleotides can also be distributed in liposomal formulations and lipofectin formulations and the like prepared by methods well known to those skilled in the art. Such 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. U.S. Patent No. 5,770,580 describes methods of gene therapy of distribution in the eye. I I The constructs of the VEGF-2 polynucleotide vector used in the gene therapy method are preferably constructs that will not migrate to the gjenome i of the host nor will they contain sequences that allow their reproduction. Suitable vectors include pWLNEO, pSV2CA T, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEF-L / V5, I pcDNA3.1. and pRc / CMV2 available from Invitrogen. Other suitable vectors would be readily apparent to those skilled in the art. i I I I Any strong promoter known to those skilled in the art can be used to direct the ^ & ^ expression VEGF-2. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; the promoter of the respiratory virus (RSV); inducible promoters, such as MMT promoter, the metallothionein promoter; I heat shock promoters; albumin promoter; the promoter of, ApoAI; i human globin promoters; viral promoters of the thymine kinase, such as the thymidine kinase promoter of Herpes Simplex; viral thymidine kinase promoters; Retroviral LTR; the b-actin promoter; and promoters of the human growth hormone. The promoters can also be the native promoter for VEGF-2. f i | Unlike other genetic therapy techniques, a major advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in cells.

Studies have shown that the DNA sequences that Ino reproduce can be introduced into the cells to provide for the production of the desired polypeptide for periods of up to six months.

The polynucleotide constructs of VEGF-2 can be distributed to the interstitial space of tissues within an animal, including the muscle, skin, brain, liver, spleen, bone marrow, lymph, blood, bones, cartilage, pancreas, kidney, bladder, stomach, intestine, testes ovary, uterus, rectum, nervous system, eye, glands and connective tissue. The eye is especially preferred. The interstitial space of the tissues comprises the fluid intracellular matrix I, mucopolysaccharide, between the reticular fibers of organ tissues, elastic fibers in the walls of the vessels or chambers, fibers of collagen or fibrous tissues, or the same matrix Within the muscle cells that form the connective tissue or in the lacuna of the bone, this is also the space occupied by the plasma, the circulation and the lymphatic fluid of the lymphatic channels. Injection into the tissues comprising these cells, they are preferably distributed to and expressed in non-divisible persistent cells, which are differentiated, although the distribution and expression can be achieved in undifferentiated or less completely differentiated cells, such as example, undifferentiated cells of the blood or fibroblasts of the skin.In vivo muscle cells are I are particularly competent for their ability to absorb and express polynucleotides.

For the injection of the naked 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 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. Of course, as one skilled in the art will appreciate, this dosage will vary according to the tissue injection site. The appropriate and effective dose of nucleic acid sequence can easily be determined by those skilled in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the intraparenteral injection route into the interstitial space of tissues, especially the eye. However, other parenteral routes, such as inhalation of an aerosol formulation particularly i for distribution to the lungs or bronchial tissues, throat or mucous membranes of the nose, may also be used. In addition, the naked DNA constructs of VEGF-2 can be distributed to arteries during angioplasty by the catheter I used in the procedure. Naked polynucleotides are distributed or released by topical administration and so-called "gene guns" or "genetics." I Such methods of distribution are known in the art.1 The constructs can also be distributed with delivery or delivery vehicles such as sequences. Viral particles, viral particles, liposomal formulations, iipofectin, precipitating agents, etc. Such methods of distribution or release are known in the art.In certain embodiments, the lyucleotide constructs of VEGF-2 are complexed in a liposomal preparation. Lipid preparations for use in the present invention include cationic (positively bound), anionic (negatively charged) and neutral preparations, however, cationic lipidomas are particularly preferred because a strong charge complex can be formed between cationic lipogenesis and polyanionic nucleic acid. Cationic liposomes have been shown to mediate the intracellular distribution of piasmidic DNA (Felgner et al., Proc. Nati Acad. 'Sci. I i USA (1987) 84: 7413-7416, which is incorporated herein by reference; MRNA (Malone c-: r al., Proc. Nati. Arad. Sci < USA (1989) 86: 6077-6081, which is incorporated herein by reference); and tiar-scripting factors: -purified urine (Debs et al., J. Biol. Chem. (1990- 255: 10189-10192, which is incorporated herein by reference), in functional form. Orthodontic liposomes are easily available, for example, the 1 iposomes of N [1,2,3- ^ * ^^ * jfefe ^ dioleyloxy) propyl] -N, N, N-triethylammonium (DOTMA) are particularly useful and are available under the Lipofectin brand, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Na ti. Acad. Sci. USA (1987; 4: 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 methods 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 DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane). preparation of DOTMA lisposomes is explained in the literature, see, for example, P. Felgner et al., Proc. Na ti.Accid. Sci. USA 84: 7413-7417, which is incorporated herein by reference.

Similar methods can be used to prepare I liposomes from 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. Such materials include phosphatidyl choline, cholesterol, 1-phe- sophatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoyl phosphatidyl I ethanolamine (DOPE), among others. These materials can also be mixed with the initial materials DpTMA and, DOTAP in appropriate relationships. Methods for producing liposomes using those materials are well known in the art. i For example, dioleoylphosphatidyl choline (DOPC),! dioleoylphosphatidyl glycerol (DOPG), and commercial dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to produce conventional liposomes, i with or without the addition of cholesterol. Thus, for example, DOPG / DOPC vesicles can be prepared by drying 50 mg of each of the DOPG and DOPC under a flow of nitrogen gas in a sonication flask. The sample is coilocada under a pump of empty during the night and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a stoppered bottle, using a Model 350 sonicator from Heat Systems equipped with an inverted cup probe (bath type) at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication I to produce multilamellar vesicles or by extrusion through nucleophore membranes to produce unilamellar vesicles of discrete size. Other methods are known and The nucleic acids are prepared using well-known methods in the technique. See, for example, Straubinger et al. , Meth ds of Immunology (1983), 101: 512-527, 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 prolonged sonication of MLVs to produce a homogenous population of unilamellar liposomes. The material to be trapped is added to a suspension of the MLV produced and then sonicated.

When liposomes containing cationic lipids are used, the dry lipid film is resuspended in one! appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris / NaCl, sonicate, I and then the preformed liposomes are directly mixed with the DNA. The liposome and DNA forit-an I very stable complex due to the binding of liposomes positively charged to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include chelation Ca2 + -EDTA (Papahadjopoulos et al., Biochim Biophys. Acta (1975) 394: 483; Wilson et al., Cell (1979) 17:77); ether injection (Dea er, D. and Bangham, A., Biochim.

Biophys. Acta (1976) 443: 629; Ostro et al., Biochem. Biophys Res. Commun. (1977) 76: 836; Fraley et al, Proc. Nati Acad. Sci. USA (1979) 76: 3348); dialysis with detergent (Enoch, H. and Strittmatter, P., Proc. Matl. Acad. Sci. USA (1979) 76: 145); and reverse phase evaporation (REV) (Fraley et al; J. Biol. Chem. (1980) 255: 10431; Szoka, F. y Papahadjopoulos, D., Proc. Nati Acad. Sci. USA (1978) 75: 145; i Schaeffer-Ridder et al; Science (1982) 215: 166), which are incorporated herein by reference.

Generally, the ratio of the DNA to the liposomes would be from about 10: 1 to about 1:10 Preferably, the ratio would be from about 5: 1 to about 1: 5. More way! i preferable, the ratio would be from about 3: 1 to 1 about 1: 3. Even more preferably] the ratio will be approximately 1: 1. US Pat. No. 5,676,954 (which is incorporated herein by reference) reports on the injection of genetic material, complexed with liposomal, cationic carriers (, in mice, US Patents Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127 , 5,589,466, 5,693,622, 5,580,859, 5,703,055, and International Publication No. WO 94/9469 (which are incorporated herein by reference) provide cationic lipids for use in the transfection of DNA in cells and mammals US Patents 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 distributing cationic DNA-lipid complexes I to mammals. are modified, ex vivo or in vivo, using a retroviral particle i containing RNA which comprises a sequence that codes for to VEGF-2 Retroviruses from which retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, splenic necrosis virus, Rous sarcoma virus, Virus of Harvey's Sarcoma, leucodis virus of birds, gibbon leukemia virus, human immunodeficiency virus I, Myeloproliferative Sarcoma Virus, and mammary tumor virus. The retroviral plasmid vector is used to transduce i, packaging cell lines to form lines of producer cells. Examples of packaging cells that can be transfected include, but are not limited to, PE501 cell lines, PA317, f-2, f-AM, PA12, T19-14X, VT-19-17-H2, fCRE, fCRIP, GPYE-86, GP + envAml2, and DAN, as described in Miller, Humah Gene Therapy 1: 5-14 (1990), which is incorporated here as a reference in its entirety. The vector can be transduced to the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and precipitation with CaP04. In an alternative, the retroviral plasmid vector can be encapsulated in a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious retroviral vector particles which include the 1 polynucleotide encoding VEGF-2. Such retroviral vector particles i can then be used, I for! Transduce eukaryotic cells, whether in vi tro or in vivo. I The transduced eukaryotic cells will express VEGF-2. In certain other embodiments, the cells are modified, ex vivo or in vivo, with the polynucleotide of the VEGF-2 contained in an adenovirus vector. The adendvirus can be manipulated to code for and express VEGF-2, and to be inactivated in terms of its ability to reproduce in a normal UTI life cycle. Expression of the adenovirus is achieved without integration of the viral DNA into the chromosome of the host cell, thereby alleviating concerns about insertional mutagenesis. In addition, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev. Respir. Dis. 109: 233-238). Finally, the gene transfer is mediated by the adenovirus has been demonstrated in a number of cases including the transfer of alpha-1-antitrispina and CFTR in the lungs of cotton rats (Rosenfeld, M. et al. (1991) Science 252: 432-434; Rosenfeld et al. (1992) Cell 58: 143-155). In addition, extensive studies for attempting to establish adenoviruses as a causative agent of human cancer were uniformly negative (Green, M. et al. (1979) Proc. Nati. Acad. Sci. I USA 75: 6606). Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Current Opinion in Genetics Devel. 3: 499-503 (1993); Rosenfeld et al. (1992) Cell 58: 143-155 (1992); Engelhardt et I I al., Human Genet. Ther. 4: 159-169 (1993); Yang et al., Nature and Genet. 7: 362-369 (1994); Wilson et al., Nature 365: 691-692 i (1993); and U.S. Patent No. 5,652,224, which are incorporated herein by reference. For example, the Ad2 adenovirus vector is useful and can be grown in human 293 S cells. These cells contain the El region of the adenovirus and constitutively express Ela and Elb, which complement it. s ^ »* ^^^ 9 ^« ^ * jA ^ * ^^ j ^^^ ------ ¿^ ^^ to the defective adenovirus providing the products of the deleted genes of the vector. In addition to Ad2 other varieties of adenoviruses (eg, Ad3, Ad5 and Ad7) are also useful in the present invention. i Preferably, the adenoviruses used in | the present invention are deficient in their reproduction. Deficient reproduction adenoviruses require the aid of an auxiliary virus and / or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express the polynucleotide of the VEGF-2 of interest which is operably linked to a promoter, but can not reproduce in most cells. Deficient reproduction adenoviruses can be deleted in one or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or Ll to L5. In other certain modalities, cells | are modified i I, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are natural defective viruses, which require auxiliary viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Mi crobiol.

Immunol. 158: 91 (1992)). This is also one of the few viruses that can integrate its DNA into cells that are not, dividing. The vectors that contain as few as | 300 I base pairs of AAV can be packaged and can be integrated, but the space for the exogenous DNA is limited to approximately 4.5 kb. Methods for producing and using such AAV 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 reproduction, encapsulation and integration into the host cell of the DNA. The polynucleotide construct of VEGF-2 is inserted into an AAV vector using standard cloning methods, such as those found in Sambrook, et al. , Molecular Cloning. A i Labora tory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including! lipofection, electroporation, calcium phosphate precipitation, etc. Suitable helper viruses include i adenovirus, cytomegalovirus, vaccinia virus or v, irus i helper. Once the packaging cells are transfected and infected, they will produce infectious viral particles, which contain the polynucleotide construct of VEGF-2. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct of VEGF-2 integrated into their genome, and will express VEGF-2. Another method of gene therapy involves operably associating the heterologous control regions and 96/29411, published on September 26, 1996; International Solitude No. WO 94/12650, published on 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) J This method involves the activation of a gene which is present in the target cells, but which is normally not expressed in the cells, or is expressed at a lower level of wanted. j Polynucleotide constructs are made, using standard methods known in the art; I which contain the promoter with the leader sequences | I flanked the promoter. The right promoters! they are described here. The leader sequence is sufficiently complementary to an endogenous sequence to allow the! homologous recombination of the leader sequence of the promoter with the endogenous sequence. The leader sequence will be sufficiently close to the 5 'end of the desired endogenous polynucleotide sequence of VEGF-2 so that the promoter is operably linked to the endogenous sequence after 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 same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second leader sequence i contain the same restriction site as the 3' end of the promoter amplified. The amplified promoter and the leader sequences are digested and ligated together. The leader sequence construct of the proton is released to the cells, either as a naked polynucleotide, or in conjunction with agents that facilitate transfection, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc. ., described in more detail! later. The leader sequence of the promoter can be released by a method, including direct injection with a needle, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are The promoter leader sequence construct is absorbed by the cells. The homologous recombination between the construct and the endogenous sequence takes place, so that an endogenous VEGF-2 sequence is placed under the control of the promoter. The promoter then directs the expression of the endogenous VEGF-2 sequence. Preferably, the polynucleotide encoding VEGF-2 contains a secretory signal I sequence 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 or any of the polynucleotide constructs described above can be used in both the i mode resulting in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct injection with a needle, systemic injection, catheter infusion, biolistic injectors, particle accelerators (eg, "gene guns"), gel foam sponge reservoirs, other reservoir or reservoir materials commercially available, osmotic pumps (for example, Alza minipumps) oral or solid pharmaceutical formulations (tablets or pills) in the form of suppositories, and decanting or topical application during surgery. For example, direct injection of plasmid precipitated with calcium phosphate in the rat liver and rat spleen or a plasmid coated with protein in the portal vein has resulted in gene expression of the external gene in the rat livers (Kaneda et al., Sci ence 243: 315 (1989)). | A preferred method of local administration is by direct injection. Preferably, a recombinant molecule i of the present invention complexed with a delivery vehicle is administered by direct injection locally into the area of the arteries1. The administration of a composition locally within the area of the arteries refers to the injection of the composition in centimeters and preferably into millimeters within the arteries. Another method of local administration is contacting a polynucleotide construct herein on or around a surgical wound. For example, a patient may undergo surgery and the polynucleotide construct may be coated on the surface of the tissue within the wound or the construct may be injected into areas of the tissue within the wound. Therapeutic compositions useful in systemic administration include recombinant molecules of the present invention complexed with a targeted delivery vehicle of the present invention. Delivery or delivery vehicles suitable for use with systemic administration comprise liposomes comprising ligands to direct the vehicle to a particular site. Preferred methods of systemic administration include intravenous injection, distribution or aerosol, oral or percutaneous delivery (topical ). Intravenous injections can be performed using standard methods in the art. The aerosol distribution can also be effected using standard methods in the art (see, for example, Stribling et al., Proc. Na ti. Acad. Sci. USA 189: 11211-11281, 1992, which is incorporated herein by reference. ). The oral distribution can be effected by completing a polynucleotide construct of the present invention with a carrier capable of resisting degradation by digestive enzymes in the intestine of an animal. Examples of such carriers include plastic capsules or tablets, such as those known in the art. The topical distribution i can be effected by mixing a polynucleotide construct of . ^ - & , ¡-tes. the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing to the skin. The determination of an effective amount of substance to be distributed may depend on a number of factors including, for example, the chemical structure and the biological activity of the substance, the age and pelt of the animal, the precise condition required by the treatment and its severity, and the administration route. The frequency of the treatments depends on a number of factors, such as the number of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and time of doses will be determined by the attending physician or veterinarian. ! The therapeutic compositions of the present invention can be administered to any animal, preferably mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cattle, horses and pigs, with humans being particularly preferred.

Utility of Nucleic Acids 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 manufacturing of DNA vectors, and for the production of diagnostic and therapeutic agents to treat human diseases. For example, VEGF-2 can be used for the cultivation of photoreceptor cells, where it is added to the conditional medium in I at a concentration of 10 pg / ml to 10 mg / ml. Fragments of the full-length VEGF-2 gene can be used as a hybridization probe for a cDNA library to isolate other genes having a high sequence similarity to that of the gene or a similar biological activity. Probes of this type generally have at least 50 base pairs, although they may have a greater number of bases. The probe 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 and promoter regions, exons and introns. An example of a separation or selection comprises isolating the coding region of the VEGF-2 gene using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to separate or select a library of human cDNA, genomic DNA or mRNA to determine which members of the library hybridize the probe. 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 skilled in the art, for example, separation and FACS sorting of the ligand (Coligan et al., Curren t Protocols an Immun., 1 (2), Chap. 5, (1991)). Preferably, expression cloning is employed where the polyadenylated RNA is prepared from a VEGF-2 responsive cell, and a cDNA library created from this RNA is divided into parts and used to transfect COS cells or other cells that Do not be sensitive or respond to VEGF-2. Transfected cells that are grown on slides are exposed to labeled VEGF-2. VEGF-2 can be labeled by a variety of means including the iodization or inclusion of a site of the receptor, the labeled VEGF-2 can be ligated by photoaffinity with the cell membrane or preparations} from -.- - k. extracts that express the receptor molecule. The crosslinked material is resolved by PAGE and exposed to an X-ray film. The labeled complex containing the VEGF-2 is then separated, resolved into peptide fragments and subjected to protein microsequencing. The sequence of I I I amino acids obtained from the microsequencing will be used to design a set of degenerate oligonucleotide probes to select a cDNA library to identify the gene encoding the putative receptor.

EXAMPLES 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, unless otherwise specified, are by weight. To facilitate the understanding of the following examples, certain methods and / or terms that are frequently encountered will be described. i "Plasmids" are designated by a preceded p! and / or followed by capital letters and / or numbers. The initial plasmids are commercially available, available to the public on an unrestricted basis, or can be constructed from available plasmids according to the published procedures. In addition, plasmids equivalent to those described are known in the art to be apparent to one skilled in the art. "Digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme that acts only on certain sequences in DNA. The different restriction enzymes used herein are commercially available and their conditions, cofactors and other reaction requirements were used as would be known to those skilled in the art. For analytical purposes, 1 mg of plasmid or DNA with approximately 2 units of enzyme was typically used in approximately 20 Fl of buffer. For the purpose of isolating DNA fragments The cleavage was carried out using the polyacrylamide gel 8 percent described by Goeddel, D. et al. , Nucleic Acids Res. 8: 4057 (1980). "Oligonucleotides" refers to a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands, which can be chemically synthesized. Such synthetic oligonucleotides do not have 5 'phosphate and thus will not bind to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. The synthetic oligonucleotide will be ligated to a fragment that has not been dephosphorylated. I "Ligation" refers to the process of forming phosphodiester bonds between two double-stranded nucleic acid fragments (Sambrook, et al., Molecular Cloning, A Labora tory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. (1989), p.146). Unless otherwise indicated, 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 otherwise stated, the transformation is carried out according to what is described by the method of Graham, F. and Vander Eb. , A., Virology 52: 456-451 (1973). l . ? £ - z & and > i a &JflftSfca Example 1 Expression Pattern of VEGF-2 in Human Tissues and Breast Cancer Cell Lines The analysis of northern electro-western blotting was performed to examine the expression levels of VEGF-2 in human tissues and cell lines. breast cancer in human tissues. Total cellular RNA samples were isolated with RNAzol ™ B system (Biotecx Laboratories, Inc.). Approximately 10 mg of the total RNA isolated from each tissue and specified breast cell line were separated on 1% agarose gel and stained on a nylon filter, (Sambrook, et al., Molecular Cloning, A Labora tory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). The labeling reaction is carried out according to the Stratage Prime-It kit with 50 ng of DNA fragment. The labeled DNA was purified with a Select-G-50 column, 5 Prime-3 Prime, Inc. (Boulder, CO). The filter was then hybridized with a full length VEGF-2 gene radioactively labeled at 1,000,000 cpm / ml in 0.5 M NaP04 and 7% SDS during the night 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 to -70 ° C overnight with an intensifying screen. A message of ^^? ^^^ to 1.6 Kd in two lines of breast cancer cell. Figure 5, lane # 4 represents a tumorigenic cell line that is independent of estrogen for growth. Also, 10 mg of total RNA was separated from 10 human adult tissues on an agarose gel and stained on a nylon filter. The filter was then hybridized with a VEGF-2 probe radioactively labeled in 7% SDS, 0.5 M NaP04, pH 7.2; 1% BSA overnight at 65 ° C. After washing in 0.2 X SSC at 65 ° C, the filter was exposed to a film for 24 days at -70 ° C with an intensifying screen. See Figure 6.

EXAMPLE 2 Expression of the Truncated Form of VEGF-2 (SEQ ID NO: 4) by In Vitro Transcription and Translation The VEGF-2 cDNA was transcribed and translated in order to determine the size of the translatable polypeptide encoded by the truncated form of the VEGF-2 and a partial I VEGF-2 cDNA. The two VEGF-2 inserts in the pBluescript SK vector were amplified with PCR with three pairs of primers, 1) forward and forward M13 primers; 2) i reverse M13 primer and VEGF primer F4; and 3) reverse M13 primer and VEGF primer F5. The sequences of those primers are as follows.

Reverse M13-2 primer: 5-ATGCTTCCGGCTCGTATG-3 '(SEQ ID NO: ll). This sequence is located upstream of the 5 'end of the VEGF-2 cDNA insert in the pBluescript vector and 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: 12). This sequence is located downstream from the 3 'end of the VEGF-2 cDNA insert in the pBluescript vector and is in antisense orientation as the cDNA insert. VEGF primer F4: 5 '-CCACATGGTTCAGGAAAGACA-3 (SEQ ID NO: 13). This sequence is located within the cDNA in VEGF-2 in an antisense orientation from bp 1259-1239, which is approximately 169 bp from the 3 'end of the high codon and at approximately 266 bp before the last I nucleotide of the cDNA. The PCR reaction with the three primer pairs produces products amplified with the T3 promoter sequence in the front of the cDNA insert. The first and third primer pairs produce PCR products i that code for the VEGF-2 polypeptide shown in SEQ ID NO: 4. The second pair of primers produces a PCR product that lost 36 amino acids from the coding sequence at the C terminus of the VEGF-2 polypeptide. t Approximately 0.5 mg of the PCR product i of this first pair of primers, 1 mg of the second pair of I primers, 1 mg of the third pair of primers were used for transcription / translation in vi tro. The in vitro transcription / translation reaction was performed in a volume of 25 Fl, using the TNTJ Reticulocyte Lysing Systems Coupled (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 amino acid mixture (less methionine) 1 mM, 4 Fl of 35 Sr-methionine ( > 1000 Ci / mmo1, 10 mCi / ml), 1 Fl RNAin inhibitor 40 U / μl, 0.5 or 1 mg of PCR products. Nuclease-free H20 was added to bring the volume to 25 Fl. The reaction was incubated at 30 ° C for hours. Five microliters of the reaction product was analyzed on an SDS gel.

PAGE with a gradient of 4-20%. After fixing in 2: 5% isopropanol and 10% acetic acid, the gel was dried and exposed to an X-ray film overnight at 70 ° F. As shown in Figure 7, the products of the I PCR containing the truncated VEGF-2 cDNA (ie, 'as described in SEQ ID NO: 3) and the cDNA without the 266 bp in the 3' untranslated region (3 '-UTR) produced the same length of the translated products, whose molecular weights were estimated, were from 30 to 40 kd (lanes 1 and 3). The cDNA without all the 3 'UTR and without the sequence coding for the terminal 36 amino acids was translated into a polypeptide with an estimated molecular weight of 36 to 38 kd (lane 2).

Example 3 Cloning and Expression of VEGF-2 Using the Baculovirus Expression System The DNA sequence encoding the VEGF-2 protein without 46 amino acids at the N-terminus, see e No.

ATCC 97149, was amplified using oligonucleotide PCR 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: 14) and contains a BamHI restriction enzyme site (in bold) and a sequence of 17 nucleotides complementary to the 5 'sequence of VEGF- 2 (No. 150-166). The 3 'primer has the sequence GAC TCT AGA TTA GCT CAT TTG TGG TCT (SEQ ID NO: 15) and contains the site of cleavage for the restriction enzyme Xbal and 18 nucleotides i ..- i »» .------- -a complementary to the 3 'sequence of VEGF-2, which includes 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 I fragment was digested with the BamHI endonuclease and Xbal and then subsequently purified on a agarqsa gel 1%. This fragment was ligated to the baculovirus transfer vector pAcGP67A (Pharmigen) at the BamHI and Xbal sites.

Through this ligation, the VEGF-2 cDNA was cloned into the frame with the baculovirus gα 67 gene signal sequence and located at the 3 'end of the signal sequence in the vector. This was 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 of the upstream sequence is "Geneclean". It was designated as F2. | The vector PRG1 (modification of vector pVL941) was used for the expression of the VEGF-2 protein using the baculovirus expression system (for a review see: Summers, MD and Smith, GE, "A Manual of I 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 Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHI, Smal, Xbal, BglII and Asp718. A site for the restriction endonuclease Xhol is located upstream of the BamHI site. The sequence between Xhol and BamHI is the same as for the vector PAcGp67A (static on the tape). The polyadenylation site of simian virus (SV) 40 was used for efficient polyadenylation. For easy selection of recombinant viruses, the E. coli beta-galactosidase gene was 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 viral sequences for the cell-mediated homologous recombination of co-transfected native 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 is then dephosphorylated using sheep 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 as V2, F2 fragment and dephosphorylated plasmid V2 were ligated with T-DNA ligase, followed by transformation of E. coli HB101 cells and the identified bacteria - containing the plasmid (pBac gp667- VEGF-2) with the VEGF-2 gene using the enzymes BamHI and Xbal.The sequence of the cloned fragment was confirmed by sequencing of the DNA.Patc 5 g of the plasmid pBac gp67-VEGF-2 was cotransfected with 1.0 mg d and commercially available linearized baculovirus ("BaculoGoldJ Baculovirus DNA", Pharmi ngen, San Diego, CA. ) using the lipofectin method (Felgner et al., Proc.Nat.Acid Sci USA 84: 7413-7417 (1987)) lmg of irus BaculoGoldJ DNA and 5mg of the plasmid pBac gp67-VEGF- were mixed 2 in a sterile well of a microtiter plate containing 50 ml of Grace li Dre serum medium (Life Technologies Inc., Gaithersburg, MD).

Subsequently, 10 ml of Lipofectin plus 90 iml of Grace's medium were added, mixed and incubated for 15 minutes at room temperature. The transfection mixture was then added by dripping to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mM tissue culture plate with 1 ml Grace's medium without serum. The plate was shaken back and forth to mix the newly added solution. The plate was then incubated for 5 hours at 27 ° C. 5 hours later the transfection solution was removed from the plate and 1 ml of Grace's inserts medium supplemented with 10% fetal sheep serum was added. The plate was placed again in an incubator and cultivation continued at 27 ° C for four days. Four days later the supernatant was collected and a plate test similar to that described was carried out! by Summers and Smith, supra. A modification of an agarose gel with "Blu Gal" (Life Technologies Inc., Gaithersburg) was used, which allowed easy isolation of blue-stained plates. (A detailed description d to a "plaque assay" can also be found in the user guide for the cultivation of insect cells and in baculovirology distributed by Life Technologies Inc., Gaithersburg, pages 9-10). Four days after serial dilution, the virus was added to the cells, the blue-stained plates were separated with the tip of an Eppendorf pipette. The agar containing the recombinant virus was then resuspended in an Eppendorf tube containing 200 ml of Grace's medium. The aga.r was removed by a brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells seeded on 35 mM disks. Four days later the supernatants of those culture discs were harvested and then stored at 4 ° C. Sf9 cells were grown 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 infections (MOI) of 1. Six hours later the medium was removed and replaced with 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 marine proteins were visualized by SDS-PAGE. and they autoradiograph. The protein of the medium and the cytoplasm of the Sf9 cells was analyzed by SDS-PAGE under reducing and reducing conditions. See Figures 8A and i 8B,! respectively. The medium was dialyzed against MES 50 m ?, pH i 5.8. Precipitates were obtained after dialysis and it was! resuspended in 100mM Na Citrate, pH 5.0J The influenza as described above (Wilson et al.

Cell 37: 161 (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 coding for 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: 16) which contains a BamHI site followed by 18 nucleotides from the start of the sequence coding for VEGF-2 from the start codon; the 3 'sequence (CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAG GCT CAT TTG TTG TCT 3') (SEQ ID NO: 17) containing complementary sequences for a Xbal site, the HA mark, the site Xhol, the last 15 nucleotides of the sequence encoding VEGF-2 (not including the high codon). Therefore, the PCR product contains a BamHI site, the coding sequence followed by an Xhol restriction endonuclease site and an HA tag fused in the frame, a translation stop high codon close to the HA mark, and an Xbal site. The DNA fragment amplified by PCR and the vector, pcDNAI / Amp, were digested with restriction enzyme BamHI and Xbal and ligated. The ligation mixture was transformed into E. coli SURE strain (Stratagene CtLoning '- < -, --iitf A < - r Systems, La Jolla, CA 92037), the transformed culture was grown on plates with ampicillin media and the resistant colonies were selected. The plasmid DNA was isolated from the transformants and examined by restriction analysis for the presence of the correct fragment. For the 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 Manual, Cold Spring Harbor Press , (1989)). The expression of the VEGF-2-HA protein was detected by radioactive labeling and an immunoprecipitation method (E. Harlow D. Lane, An tibodies: A Labora tory Manual, 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 harvested and the cells were used with detergent (RIPA buffer (150 m NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson et al., Cell 37: 161 (1984)) Both of the cell lysate and the culture medium were precipitated with an HA-specific monoclonal antibody.The precipitated proteins were analyzed on SDS-PAGE gels at 15 ° C. %.

Exemplary 5 Construction of carboxy terminal Amino terminal suppression mutants To identify and analyze biologically active VEGF-2 polypeptides, a panel of VEGF-2 deletion mutants was constructed using the expression vector pHE4a. 1. Construction of VEGF-2 T103-L215 in pHE4 To allow targeted amplification for the Polymerase Chain Reaction and subcloning of VEGF-2 T103-L215 (amino acids 103 to 215 in Figure 1 or SEQ ID NO: 2) in the E. coli protein expression vector, pHE4, two oligonucleotide primers complementary to the desired reaction of VEGF-2 were synthesized with the following base sequence: 'primer (Nde I / START and 18 nt of the coding sequence): 5' -GCA GCA CAT ATG ACA GAA GAG ACT ATA AAA-3 '(SEQ ID NO: Primer 3' (Asp718, HIGH, and 15 nt of coding sequence): 5 '-GCA GCA GGT ACC TCA CAG TTT AGA CAT GCA-3' (SEQ ID NO 19! The 5 'primer described above (SEQ ID NO: 18), incorporates a restriction site of Ndel and the 3 'primer described above (SEQ ID NO: 19), incorporates an Asp718 restriction site The 5' primer (SEQ ID NO: 18) also contains an adjacent ATG sequence and the frame with the region coding for VEGF-2 to allow translation of the cloned fragment in E. coli, while the 3 'primer (SEQ ID NO: 19) contains a codon of high (preferably used in E. coli) adjacent and the frame with the coding region of VEGF-2 which ensures the correct termination of the translation in E. coli The Polymerase Chain Reaction was performed using standard conditions well known to those skilled in the art and the nucleotide sequence for mature VEGF-2 (aa 24-419 in SEQ ID NO: 2) for example, as constructed in Example 3 as a standard. The resulting amplicon was digested by restriction of Ndel and Asp718 and subcloned into Ndel / Asp718 digested with the expression vector pHE4a. 2. Construction of VEGF-2 T103-R227 in pHE4 To allow amplification directed by the Polymerase Chain Reaction and subcloning of i VEGF-2 T103-R227 (nucleic acids 103 to 227 in Figure 1 SEQ ID NO: 2) into the E protein expression vector. coli, pHE4, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following base sequence: '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: 20) 3 'primer (Asp718, HIGH, and 15 nt of the coding sequence): 5' -GCA GCA GGT ACC TCA AGC TCT AAT AAT GGA-3 '(SEQ ID NO: 21) In the case of the primers described above, a restriction site of Ndel or Asp718 was incorporated into the 5 'primer and the 3' primer, respectively. The 5 'primer (SEQ ID NO: 20) also contains an ATG sequence adjacent to and in frame with the region encoding p = .ra VEGF-2 to allow translation of the fragment cloned into E. coli, whereas the 3 'primer (SEQ ID NO: 21) contains a codon of high (preferably used in E. coli) adjacent to and in frame with the coding region of VEGF-2 which ensures the correct termination of the translation. coli. The Polymerase Chain Reaction was performed using standard conditions well known to those skilled in the art and the nucleotide sequence for the ... -.-- && amp; -.--. - mature VEGF-2 (aa 24-419 in SEQ ID NO: 2) for example, as constructed in Example 3 as a standard. The resulting amplicon was digested by restriction with Ndel and Asp718 and subcloned into Ndel / Asp718 digested with the protein expression vector pHE4a. 3. Construction of VEFG-2 T103-L215 in pA2GP In this illustrative example, the plasmid release vector pA2 GP is used to insert the cloned DNA encoding the N-terminal and C-terminal deleted VEGF-2 protein (amino acids 103- 215 in Figure 1 or SEQ ID NO: 2), in a baculovirus to express the N-terminal and C-terminal deleted VEGF-2 protein, using a baculovirus leader and standard methods as described in Summers et al. , A Manual of Methods for Baculovirus Vectors and Insect Cell Cul ture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555, (1987)). This expression vector contains the strong polyhydrin promoter of Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader) of baculovirus gp67 protein and convenient restriction sites such as BamHI, Xbal and Asp718. The polyadenylation site of simian virus 40 ("SV40") was used for efficient polyadenylation. For easy selection of recombinant viruses, the plasmid contains the E. coli beta beta-galactosidase gene or the control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhex gene. rina The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with the native viral DNA to generate viable viruses that express the cloned polynucleotide. Many other baculovirus vectors could be used instead of the previous vector, such as pAc373 , pVL941 and pAcIMl, as one skilled in the art would readily appreciate, as long as the construct provides the appropriate localized signals for transcription, translation, secretion and the like, including a signal peptide and an intraframe AUG as required. Such vectors are described, for example, in Luckow et al. , Virology 27/9: 31-39 (1989). The cDNA sequence coding for the VEGF-2 protein without 102 amino acids at the N-terminus and without 204 amino acids at the C-terminus of Figure 1, was amplified using PCR oligonucleotide primers corresponding to the 5 'sequences 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 BamHI (in bold) followed by 1 separator of a nt to remain in box with the peptide ? To signal provided by the vector, and 17 nt of the bases of the coding sequence of the VEGF-2 protein. The 3 'primer has the sequence 5' -GCA GCA TCT AGA TCA CAG TTT AGA CAT GCA-3 '(SEQ ID NO: 23) which contains the restriction site Xbal (in bold) followed by a high codon and nucleotides complementary to the coding sequence: 3 '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 fragment was then digested with the endonucleases Ba-nHI and Xbal and then purified again on a 1% agarose gel. This fragment was ligated to the pA2 GP baculovirus transfer vector (Supplier) at the BamHI and Xbal sites. Through this ligation, the VEGF-2 cDNA representing the N-terminal and C-terminal deleted VEFG-2 protein (amino acids 103-215 in Figure 1 or SEQ ID NO: 2) was cloned in frame with the signal sequence of the baculovirus GP gene and located at the 3 'end of the signal sequence in the vector. This was 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 • ^ tmA? U 26 nt of the bases of the coding sequence of the VEGF-2 protein. The 3 'primer has the sequence 5' -GCA GCA TCT AGA ACG TCT AAT AAT CGA ACTG AAC-3 '(SEQ ID NO: 25) containing the Xbal restriction site (in bold) followed by a high codon and nucleotides complementary to the 3 'coding sequence of VEGF-2. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit ("Geneclean", BIO 101, Inc. La Jolla, CA The fragment was then digested with the BamHI and Xbal endonucleases and then purified again on a gel of 1% agarose This fragment was ligated to the vector of baculovirus pA2 GP transfer (Supplier) at the BamHI and Xbal sites, through this ligation, the VEGF-2 cDNA representing the VEFG-2 protein deleted N-terminal and C-terminal (amino acids 103-227 in Figure 1 or SEQ ID NO: 2) was cloned in frame with the signal sequence of the baculovirus GP gene and located at the 3 'end of the signal sequence in The vector was 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 or amplifier (Boshart et al., Cell 41: 521-530 (1985)). Multiple cloning sites, for example, with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3N intron, the polyadenylation signal and termination 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 [No. Access ATCC 37146]. Both plasmids contain the mouse DHFR gene under the control of the SV40 large promoter. Chinese hamster ovarian cells or others 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 methotrexate. The amplification of DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, for example., Alt, F.W., Kellems, R.M., Bertino, J.R., and Schimke, R.T., 1978, J. Bi ol. Chem. 253: 1351-1310, Hamlin, J.L. and Ma, C. 1990, Biochem. et Biophys. Minutes 1097: 101-143, Page, M.J. and Sydenham, M.A. 1991, Biotechnology 9: 64-68). Cells growing in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of the amplification of the DHFR gene. If a second gene is linked to the DHFR gene it is usually quantified and overexpressed. This is the state of the art to develop cell lines that contain more than 1,000 copies of the genes. Subsequently, when methotrexate is removed, the cell lines contain the amplified gene integrated into the chromosomes. Plasmid pCl contains the expression of the gene of interest in a strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, March, 1985: 438-4470) plus a isolated fragment of the amplifier of the human cytomegalovirus (CMV) immediate early gene (Boshart et al., Cell 41: 521-530 1985). Downstream of the promoter are the following uric restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Pvul and Nrul. Behind these cloning sites the plasmid contains high translation codons in the three reading frames followed by the 3N intron and the polyadenylation site of the rat preproinsulin gene. Other more efficient promoters can also be used for expression, for example, the human b-actin promoter, the SV40 early and late promoters or the long terminal repeats of other viruses, for example, HIV and HTLVI. For polyadenylation of the other mRNA signals, for example, human growth hormone or globin genes can also be used. Stable cell lines containing a gene of interest integrated into the chromosomes can also be selected after 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, eg, G418 plus methotrexate The plasmid pCl is deferred with the restriction enzyme BamHI and then dephosphorylated using intestinal intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel. The DNA sequence coding for VEGF-2, Accession No. ATCC 97149, was constructed by PCR using primers corresponding to the 5 'and 3' ends of the gene ^^^ j ^ H as a protein signal sequence.

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: 2 in pC4Sig, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following base sequence. 'primer (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: 28) 3' primer (Xba I, HIGH, 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: 29) The Polymerase Chain Reaction was performed utilizing standard conditions well known to those skilled in the art and the nucleotide sequence for mature VEGF-2 (aa 24-41) for example, as constructed in Example 3 as a standard. The resulting amplicon was digested by restriction of BamHI and Xbal and subcloned into the pC4Sig vector digested with BamHI / Xbal. I - »- # t- --- a- *« - »7. Construction of pC4SigVEGF-2 T103-R227 To allow the amplification directed by the Polymerase Chain Reaction and subcloning of the .VEGF-2 T103-L215 (amino acids 103 to 227 in Figure 1 or SEQ ID NO: 2 in pC4Sig, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following sequence of bases. 'primer (Bam Hl and 26 nt of the coding sequence): 5' -GCA GCA GGA TCC ACA GAA GAG ACT ATA AAA TT | T GCT GC-3 '(SEQ ID NO: 30) 3' primer (Xba I, HIGH, and 21 nt of the coding sequence): 5 '-GCA GCA TCT AGA TCA ACG TCT AAT AAT GGA ATG AAC- 3' (SEQ ID NO: 31) The Polymerase Chain Reaction. was performed using standard conditions well known to those skilled in the art and the nucleotide sequence for mature VEGF-2 (aa 24-419) for example, as constructed in Example 3 as a standard. The resulting amplicon was digested by restriction of BamHI and Xbal and subcloned into the pC4Sig vector digested with BamHI / Xbal.

In the case of the 5 'primer described above, a BamHI restriction site was incorporated, while in the case of the 3' primer an Asp718 restriction site was incorporated. The 5 'primer also contains 6 nt, 20 nt of the sequence coding for VEGF-2, and an adjacent ATG sequence and in frame with the coding region of VEGF-2 to allow translation of cloned fragment in E. coli, whereas the 3 'primer contains 2 nt, 20 nt of the sequence encoding VEGF-2, and a high codon (preferably used in E. coli) adjacent to and in frame with the region coding for VEGF-2 , which ensures the correct translational completion in E. Coli. The Polymerase Chain Reaction was performed using standard conditions well known to those skilled in the art and the nucleotide sequence for VEGF-2 (aa 24-419) as constructed, for example, in the Example 3 as a pattern. The resulting amplicon was digested by restriction with BamHI and Asp718 and subcloned into the pC4 expression protein vector digested with BamHI / Asp718. This construct was designated pC4VEGF-2 M1-M263. 9. Construction of pVEGF-2 M1-D311 In this illustrative example, the cloned DNA that i codes for the protein M1-D311 VEGF-2 (amino acids lj-311, in Figure 1 or SEQ ID NO: 2) was inserted into the vector ^ - ^ fc plasmid pC4 for the expression of the C-terminal deleted VEGF-2 protein. To allow for amplification directed by the Polymerase Chain Reaction and subcloning of the M1-D311 of VEGF-2 into an expression vector, pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following base sequence: Primer 5 '5' -GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3 '(SEQ ID NO: 34) Primer 3' 5 '-GAC TGG TAC CTT ATC AGT CTA GTT CTT TGT GGG G-3' (SEQ ID NO: 35) In the case of the 5 'primer described above was incorporated a restriction site BamHI, 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 sequence coding for VEGF-2, and an adjacent ATG sequence and the frame with the region encoding VEGF-2 to allow translation of the fragment cloned in E. coli while the 3 'primer contains 2 nt, 20 nt of the sequence coding for VEGF-2, and a codon of high (preferably used in E. coli) adjacent to and in frame with the region coding for VEGF- 2 which ensures the correct termination in E. coli The Polymerase Chain Reaction was performed standard conditions well known to those skilled in the art and the nucleotide sequence for mature VEGF-2 (aa 24-419) as constructed, for example, in the Axis 3 as a Pattern. The resulting amplicon was digested by restriction with BamHl and Asp718 and subcloned into expression vector of pC4 protein digested with BamHl / Asp718.

. Construction of pVEGF-2 M1-Q367 In this illustrative example, the cloned DNA encoding the protein M1-D311 VEGF-2 (amino acids 1-311, SEQ ID NO: 2) was inserted into the plasmid vector pC4 for the expression of the C-terminal deleted VEGF-2 protein. To allow for amplification directed by the Polymerase Chain Reaction and subcloning of the M1-D311 of VEGF-2 into an expression vector, pC4, two oligonucleotide primers complementary to the desired region of VEGF-2 were synthesized with the following base sequence: Primer 5 '5' -GAC TGG ATC CGC CAC CAT GCA CTC GCT GGG CTT CTT CTC-3 '(SEQ ID NO: 36) 3' 5 'Primer -GAC TGG TAC CTC ATT ACT GTG GAG TTT CTG TAC ATT C-3' (SEQ ID NO: 37) In the case of the 5 'primer described above incorporated a BamHI restriction site, 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 sequence coding for VEGF-2, and an adjacent ATG sequence and the frame with the region encoding VEGF-2 to allow translation of the fragment cloned in E. coli while the 3 'primer contains 2 nt, 20 nt of the sequence encoding VEGF-2, and a stop codon (preferably used in E. coli) adjacent to and in frame with the region coding for VEGF- 2 which ensures the completion of the correct translation in E. coli. The Polymerase Chain Reaction was performed standard conditions well known to those skilled in the art and the nucleotide sequence for mature VEGF-2 (aa 24-419) as constructed, for example, in Example 3 as a standard . The resulting amplicon was digested by restriction with BamHl and Asp718 and subcloned into pC4 protein expression vector digested with BamHI / Asp718. This construct was designated pC4VEGF-2 M1-Q267.

Example 6 Method Used in Genetic Therapy for the Production VEGF-2 Polypeptide In Vivo I The standard DNA for the production of ^ RNm i that codes for VEGF-2 was prepared according to the »---- ..» .---- > ----. - S? F ^ jée standard recombinant DNA methodology. Endotoxin-free and sterile oligonucleotides were diluted with Balanced Salt Solution (BSS, Alcon, Fort Worth) to have the same pH and electrochemical concentration as the aqueous humor and vitreous humor of the eye, Emalphor EC620 (2.5%, GAF) was added. Corp.) (Bursell et al. (1993) J. Clin. Invest. 92: 2872-2876), a petroleum product, to change viscosity and aid in release properties. concentrations in the range of 0.1 μM-100 μM. The volume distributed is between 1 μM and 1 ml depending on the volume Of the eye. The intubated patient was anesthetized with fluoran. The face and eyes were prepared by scrubbing with betadine and covering in the usual sterile manner. The sterile polynucleotide was injected with a .33 gauge needle into a sterile syringe in the posterior limbus (pars plana) through all the thickness of the vitreous sclera. No closing suture was required unless there was a leak. Antibiotic drops containing gentamicin or erythromycin ointment were applied to the surface of the globe in the fissure and eyelid several times a day until the wound was completely closed. The frequency of the injection fluctuates from every third day I 'or once every six months or less, depending on the severity of the disease process, the degree of interocular inflammation, the character of the vehicle (ie, slow release characteristics), and the eye's tolerance to injections. Follow-up checks are needed in the short and long term for possible detachment of the retina with injections. The eye after dilatation was verified by signs of inflammation, infection, and growth of photoreceptors by direct and indirect ophthalmoscopy to see the retina and the fundus. Verification can be as frequent as daily in cases where premature infants are in danger of retinal detachment. The frequency of verification will decrease with the resolution of the disease. The patient is treated weekly with infraocular injections of polynucleotide suspended in the appropriate veniculum (BSS, Emanfour) at concentrations within the range of 0.1 to 100 μM. This treatment can be supplemented with systemic release of polynucleotide (i.e., intravenous, subcutaneous or intramuscular) from 2 to 5 times a day to once a month.

Example 7 Method of Treatment Used in Genetic Therapy - Ex vivo Homologous Recombination Photoreceptor cells were obtained from a subject by biopsy. The resulting tissue was placed in DMEM p 10% fetal sheep serum. The fibroblasts that grew exponentially or in initial stationary phase were fertilized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension was removed for counting, and the remaining cells were subjected to centrifugation. The supernatant was aspirated and the pellet was resuspended in 5 ml of electroporation buffer (HEPES mM pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 nM Na2HP04, 6 mM dextrose). The cells were centrifuged, the supernatant was aspirated and the cells were resuspended in electroporation buffer with a content of 1 mg / ml of acetylated serum albumin. The final cell suspension contains approximately 3 × 10 6 cells / ml. Electroporation should be done immediately after resuspension. The plasmid DNA was prepared according to standard techniques. To construct a plasmid to direct it to the VEGF-2 site, plasmid pUC18 (MBI Fermentas, Amherst, NY) was digested with HindIII. The CMV promoter was amplified by PCR with a Xbal site at the 5 'end and a BamHI site at the Extreme gg 3 '. Two sequences not coding for VEGF-2 were amplified via PCR: a sequence that does not code for VEGF-2 (fragment 1 of VEGF-2) was amplified with a HindIII site at the 5 'end and an Xba site on the 3 'end; the other sequence that does not code for VEGF-2 (fragment 2 of VEGF-2) was amplified with a BamHI site at the 5 'end and a HindIII site at the 3' end. The CMV promoter and the VEGF-2 fragments were digested with the appropriate i I enzymes (CMV-Xbal and BamHI promoter, VEGF-2-XbaI fragment 1, VEGF-2-BamHI fragment) and ligated together. The resulting ligation product was digested with HindIII, and ligated with the pUC18 plasmid digested with HindIII. The plasmid DNA was added to a sterile cuvette with an electrode space of 0.4 cm (Bio-Rad). The final DNA concentration is generally at least 120 μg / ml. 0.5 ml of the cell suspension (containing about 1.5X106 cells) was then added to the cuvette, and the cell suspension and DNA suspensions were mixed gently. The electroporation was carried out with a Gene-Pulser device (Bio-Rad). Capacitance and voltage they were set at 960 μF and 250-300 V, respectively. When the voltage increases, cell survival 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 can be observed. The electroporated cells were kept at room temperature for about 5 min and the content of the coating was then gently stirred with a sterile transfer pipette. The cells were added directly to 10 ml of preheated nutrient medium (DMEM with 15% sheep serum) in a 10 cm dish and incubated at 37 ° C. The next day, the media was aspirated and replaced with 10 ml of fresh media and incubated for an additional 16-24 hours. The modified photoreceptor cells were then injected into the host. Photoreceptor cells now produce a protein product.

EXAMPLE 8 Activity of VEGF-2 in Retinal Cells The retina has proven to be an advantageous experimental model for studying the role of intrinsic and extrinsic factors in the regulation of the development of neuronal and non-neuronal cell types of a more primitive neuroepithelial cell. The differentiated retina is composed of seven types of cells: sensory (rods and photoreceptor cones), glia (Müller cells), and two types of cells. --uMi ^ B neurons, interneurons (horizontal, bipolar and amacrine), and projection neurons (ganglicnares cells) (for a review see Dowling, 1987). The development of different cell types in the retina does not occur in a synchronized manner with most cones, and the development of horizontal ganglion cells; before birth (for a review see Altshuler et al., 1991, Harris, 1991, Reh, 1991). In contrast, the difference of a majority of the rods, the primary cell type in the rat retina, occurs postnatally. Clonal progeny analysis of retinal precursor cells has shown that progenitor cells can produce various combinations of retinal cell types indicating that progenitors are totipotent or multipotent depending on the developmental age examined (Turner and Cepko, 1987; Turner et al. , 1990; Wetts and Fraser, 1998). In addition, findings from studies both in vivo and in vi tro demonstrate that the final phenotype of retinal cells depends largely on the lineage, which suggests that the changing microenvironment of the retina has a role in the cellular potential determination of progenitor cells. ! oras, as well as the differentiated phenotype of the progene (Watanabe yi Raff, 1990, 1992; Harris, 1991; Reh, 1991; Ezzeddine et al. 1997).

In vi tro, retinal cell proliferation and differentiation is regulated by a variety of factors; for example, FGF-2 (Hicks and Courtois, 1992), CNTF (Ezzeddine et al., 1997, Fuhrmann et al., 1995), LIF (Ezzeddine et al., 1997), TGF (Lilleen and Cepko, 1992). ), retinoic acid (Kelly r-? et al., 1994) and EGF (Lillien, 1995). Recently, Yang and Cepko (1996) have identified and characterized the expression pattern of VEGFR-2 / FLK-1 in developing and adult retina. i i VEGFR transcripts are first detected in Eli.5 in association with the development of the retinal vasculature and with the central region of the neural retina. At the E15 development day, the expression of VEGFR-2 extends to the periphery of the retina consisting of the outward gradient of the retinal development (Young, 1985; La Vail et al., 1991). The expression of VEGFR-2 was found largely in the ventricular zone during the perinatal period when neurogenesis is at its peak and a large number of postmitotic neurons are forming. As shown below, the greatest effect of VEGF in vi is during the initial development and involves the proliferation of multipotent progenitor cells due to the level of BrdU and the number of photoreceptor and amacrine cells increased. VEGF-2 improved the proliferation of retinal cells derived from E15 embryos and the magnitude of the response increased with age. The initial proliferative response to the administration of VEGF-2 was not effected by the CNTF. However, CNTF inhibited the increase induced by VEGF-2 at the rhodopsin protein level.

Experimental Procedures Animals. Pregnant, synchronized animals were obtained from Harlan Sprague-Dawley (Indianapolis, IN). All procedures related to the animals were conducted in strict compliance with the approved institutional protocols and in accordance with the regulations and animal care and use described in the Guide for the Use and Complaint of Laboratory Animals (NIH publication No. 86-23, 1985).

Retinal crops. The retinal tissues were obtained from late embryonic or neonatal rats. The dissociated primary cells were prepared by incubating the tissue in 0.25% trypsin for 6 min at 37 ° C. after inactivation of trypsin by a 5 min incubation in growth medium (F12: Dulbecco's modified Eagle's medium (DMEM) containing 1% fetal bovine serum, 1% hormonal supplements (N2, Bottenstein, 1983 ), 1% glutamine and 0.5% penicillin-streptomycin (10,000 units / ml and 10 mg / ml, respectively, Gibco, Grand Island, NY), containing 50 μg / ml deoxyribonuclease type I (Sigma, St Louis, MO), the tissue fragments were repeatedly passed through a Pasteur pipette with a restricted tip of a diameter of approximately 1 mm. Dissociated cells were harvested by centrifugation (800xg, 5 min) and resuspended in growth medium. The cells were seeded in 96-well plates precoated with poly-L-lysine (50 μg / ml, Sigma) and laminin (10 μg / ml Gibco), at a density of 425 cells / mm2 unless otherwise stated. The cultures were gradually diverted to growth medium without serum by changing one half of the medium each day. The trophic factors were replenished with each change of medium, Hypocampic Astrocytes. Cultures purified from astrocytes were prepared from rat hippocampus using a previously described method (Greene et al., 1998). Immunohistochemistry with Rhodopsin and Cell Count Procedures. For immunohistochemical staining, the cultures were fixed overnight for 4% formaldehyde with a content of 4% sucrose. For staining with rhodopsin and syntaxin, the cultures were permeabilized with 0.05% saponin in PBS for 30 minutes. Binding of non-specific IgG was inhibited by incubating the cells in PBS containing 5% horse serum and 2% BSA for 3 h at room temperature. The cultures were then incubated overnight at 4 ° C with anti-rhodopsin (1: 10,000, Rho 4D2, Dr, Molday, University of British Columbia) or anti-syntaxin 1: 10,000, Sigma) diluted in PBS with a content of 5% horse serum and 2% BSA (Molday, 1989). After removal of the primary antibody, the cultures were incubated with anti-mouse biotinilide antibody (1: 2,500) for 90 min. The avidin-biotin-peroxidase complex, diluted 1:50 in PBS containing 5% horse serum and 2% BSA was then added for 60 minutes. To visualize the bound peroxidase, diaminobenzidine was used at a final concentration of 0.4 mg / ml in a 0. IM acetate buffer containing 2.5% nickel sulfate. The number of immunopositive cells per well was determined by the labeled cells in an area representing 11% of the total surface area of the well and then corrected for the total surface area. Immunohistochemistry of BrdU. The retinal cells were incubated with BrdU for 4 h and subsequently washed i twice with PBS. Then a growth medium was added again to the cultures, which were then kept in place for several time intervals. At the end of the operation period, the cultures were fixed and stained. Immunohistochemically to determine the incorporated BrdU according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN). ELISA with Rhodopsin and GFAP. For the rhodopsin ELISA, the cultures were rinsed with PBS and fixed overnight with 4% paraformaldehyde with a 4% sucrose content. The cultures were then rinsed with PBS and waterproofed with 0.05% saponin in PBS i stopped adding 2M H2SO4 and the amount of product formed was quantified by measuring the absorbance at 450 nm. The absorption of the white reagent fluctuated from 0.1 to 0.15 and the indicated values were not subtracted. The ELISA with GFAP was conducted essentially as described previously (Greene et al., 1998). ^^ g ^ Cell survival test based on measurements of Calcein AM. At the end of the incubation period the cultures were rinsed once with Ham's F-12. The calcein AM was added to a final concentration of 2 μM in 100 μl of Ham's F-12 and the cultures were incubated for 60 min at 37 degrees C. At the end of the incubation period the cultures were rinsed. Absorbance was determined at 530 nm on an ELISA plate reader. Absorption of high affinity GABA. The level of absorption of high affinity GABA was determined as described previously (Greene et al., 1998). Incorporation of [3 H] Thymidine. The cultures were treated with trophic factors for 24 h and lasted for the last 4 h, the cells were labeled with [3 H] thymidine, at a final concentration of 0.33 μM (25 Ci / mmol, Amersham, Arlington Heights, IL). Incorporated [3H] thymidine was precipitated with ice-cold 10% trichloroacetic acid for 24 h. Subsequently, the cells are rinsed with ice-cold water. After analysis in 0.5 M NaOH, the used ones and the rinses with PBS (500 ul) were collected and counted. & ^^ A & j gt? * »& ¡^ ^ ez ^ RESULTS The regulatory role of VEGF on the development of photoreceptor cells was initially investigated using derived cultures or animals 1 day after birth (PN1) . Previous reports have shown that multipotent progenitors are present during this developmental period and retain their ability to differentiate into photoreceptor cells as well as other types of retinal cells in vi tro ((Marrow et al., 1998) Treatment with VEGF-2 or VEGF -1 (R and D Systems, Minneapolis, MN) induces a dose-dependent and time-dependent increase in the level of rhodopsin protein in retinal cultures (Figures 14A) .The time course of the VEGF-induced increase in rhodopsin is relatively Slowly, consistent with the known developmental profile of photoreceptor cells, days after treatment, an increase of 25-40% in the rhodopsin protein was observed with 10 to 100 ng / ml of VEGF-2. At 9 months of treatment, these same concentrations of VEGF-2 produced an increase of 200-250% in the rhodopsin protein, and, in those latter time points, VEGF concentrations were as low as or 1 g / ml significantly increased rhodopsin levels. Changes in the amount of rhodopsin may reflect changes in the level of protein expression or changes in the number of photoreceptor cells or both. For rhodopsin and calcein emission reflected an increase in the number of photoreceptor cells, cultures, were treated with VEGF-1 or VEGF-2 for 9 days and then stained immunohistochemically to determine rhodopsin. To quantify the effects of VEGF treatment, cell counts were made. The number of cells immunopositive to rhodopsin was increased as a function of concentration with the response that had an EC50 value that had a value of 0.25 and 1.5 ng / ml for VEGF-1 and VEGF-2, respectively (Figure 15 ). At a saturating concentration of VEGF-2, an increase of 2.4 times the number of rods was observed. In addition, the response is stable in the presence of VEGF-2 concentrations as high as 100 ng / ml suggesting that VEGF-2 does not readily induce a desensitization of the biological response. The response to the dose observed with VEGF-1 is similar to that obtained with VEGF-2 which is consistent with the results of the ELISA with rhodopsin. The mechanism by which VEGF-2 induces an increase in the number of photoreceptor cells may imply an increase in the proliferation of precursor cells, greater survival of the differentiated photoreceptor cells, and / or redirection of the rod lineage pathway. To investigate whether VEGF-2 is mitogenic for retinal cells, the cultures were treated with factors 4h after culturing and then I were subsequently labeled with BrdU, after 24, 48 or 72 h. At the end of the final labeling period (72 h), the cultures were fixed and the incorporated BrdU was detected immunohistochemically. No significant increase in the number of BrdU-labeled cells was observed until 48 h after treatment, when 10 ng / ml of VEGF-2 or VEGF-1 induced an increase of 2 to 3 times (Figures 16A and 16B), respectively ). The EC50 value for the response was calculated as 1 ng / ml. The time point of 48 h seems to be close to the maximum since after a 72 h incubation, the BrdU incorporation level had a decline in the cultures treated with VEGF regardless of the concentration. However, this decline in the number of immunopositive cells is not specifically related to the administration of VEGF. The basal level of BrdU incorporation decreased from 1300 to 700 immunopositive cells per well, suggesting that a general loss in the proliferative activity of the retinal progenitor cells occurred during this period of time. In spite of the total proliferative activity low in the cultures at the final time points, the administration of VEGF still resulted in a 2-fold increase in the number of cells marked with BrdU. Similar results were obtained using [3H] thymidine incorporation (Figure 16C). To better characterize a role of VEGF during I the initial in vitro culture period, the effect of delaying the addition of the factor to the cultures was examined. Level of rhodopsin protein The initial addition of VEGF was made 4, 24 or 48 h after cultivation and the cultures were subsequently maintained for 9 days and then prepared for the ELISA with rhodopsin. 2 o-VEGF-1 as a function of the time elapsed between the cell isolation and the initial addition of the factors is described in Figures 17A and 17B, respectively The addition of factors within 4 h of the cell culture gave as This resulted in a 3-fold increase in the level of rhodopsin, however, the delay in the initial treatment with VEGF 24 or 48 h resulted in the reduction of the maximum response by 28 and 43%, respectively. In addition, after a delay of 48 h, only treatment with 100 ng / ml of VEGF-2 induced a significant increase in the rhodopsin content. Suggesting in this way that the proliferative effect that VEGF is having on retinal cells is developmentally restricted and involves the proliferation of progenitors of photoreceptors. The possibility that VEGF affects other types of retinal cells that are postnatal at birth, for example Amacrine and Muller cells, was also investigated.

The morphology of amacrine cells, I identified based on their expression of syntaxin, was examined. (The data is not shown). Treatment with VEGF induced a dose-dependent increase in the number of cells immunopositive to syntaxone with 10 ng / ml by inducing a number of endothelial cells, immunopositive cells, in retinal cultures (data not shown). To better characterize the development pattern of the VEGF response, retinal cells were isolated at different developmental stages, and the mitogenic response to VEGF-2 was quantified 48 h later by labeling the cultures with [3 H] thymidine. In addition, as previously noted, differentiation of photoreceptor cells in vi tro is dependent on density, the effect of culture density on the response to VEGF was also investigated. When the cultures were derived from E15 animals and cultured at a density of 212 cells / mm, the basal level of [3 H] thymidine incorporation is 1589 ± 94 dpm / well and treatment with VEGF-2 induced a maximum increase in 50% (Figure 19A). In contrast to the response to the dose observed with Pl cultures where saturation occurs at 10 ng / ml, the proliferative response in E15 cultures is saturated at a concentration of 1 ng / ml. In addition, there is an inverse relationship between the culture density and the mitogenic response to VEGF-2. At a density of 318 cells / mm2, a deviation to the left was noted in the dose response curve with higher concentrations of 1 ng / ml causing a desensitization of the response. At the highest density tested (425 cells / mm2), the retinal cells do not respond to VEGF-2. It is interesting to note that FGF-2 (10 ng / ml), which had a biological activity similar to that of VEGF-2 in Pl cultures (see below), inhibited the proliferation of E15 cultures in more than 62 % in higher density crops (data not shown). In cultures derived from E20 animals the basal level of incorporation of [-3H] thymidine at a culture density of 212 cells / mm2 is 3361 ± 192 and the level of stimulation of [3H] thymidine incorporation with treatment with VEGF- 2 is generally higher, fluctuating from 80 to 100%, at the lowest culture densities (Figure 19B). There was still a trend that VEGF-2 has less of an effect on crops grown at higher density. However, the inhibitory effect is much less pronounced. For Pl, where the basal level of [3H] thymidine incorporation is 478 ± 36, there is a deviation to the left in the dose response with saturation occurring at 10 ng / ml and the degree of increase is greater, in the 300% interval (Figure 19C). In addition, there is no discernible effect of the culture density on the response to VEGF-2. To characterize more fully the i response of the rod or progenitor cells of rods, the effect of EGF was compared. FGF-2 or TGFß-1 has on the number of retinal cells and on the level of rhodopsin protein with that achieved with VEGF-2. EGF, a mitogen for several cell types, induces a 31% increase in the number of retinal cells with a saturating response at 1 ng / ml and remaining stable up to 100 mg / ml (Figure 20A). However, there is no concomitant increase in the level of rhodopsin protein in cultures treated with EGF (Figure 20B). FGF-2 in a concentration range of 1-100 ng / ml induces a small increase (13%) in the number of retinal cells. In addition, FGF-2, which activates a number of FGF receptors, induces an increase in the level of rhodopsin protein. An increase of 45% in the level of rhodopsin is observed with FGF-2 concentrations as low as 1 ng / ml, resulting in an EC50 value for the response in the 0.5 ng / ml interval. Treatment with TGFβ-1 results in a decrease in both the number of retinal cells and the level of rhodopsin protein. To a At a concentration of 0.1 ng / ml, TGFβ-1 decreases calcein expression and rhodopsin protein level by 40% and 90%, respectively. The results of the i BrdU labeling experiments demonstrated that VEGF-2 increases the proliferation rate of the retinal progenitor cells. Since it is thought that the developmental pathway of the cells When photoreceptors are independent of the lineage and thus under the regulation of environmental factors (Ezzeddine ZD et al., 1997), VEGF can also modulate the development of photoreceptor cells at additional downstream sites. It has been previously determined that CNTF inhibits the differentiation of photoreceptor cells relatively late in their developmental pathway by redirecting their phenotype towards the bipolar cell lineage. To investigate the potential interaction of the two factors by co-treatment of retinal cultures with VEGF-2 at a concentration that is saturating for the induction of photoreceptor cells and various concentrations of CNTF. The increase in rhodopsin protein induced by VEGF-2 is inhibited by CNTF in a dose-dependent manner (Figure 21A). The inhibitory response had an IC50 value of 0.4 ng / ml and treatment with 100 ng / ml of CNTF resulted in complete inhibition of a VEGF-2 response. However, treatment with CNTF did not alter the total number of retinal cells in the cultures (Figure 21B). To determine whether the inhibitory effect of CNTF is an early or late event, the effect of coadministration of CNTF on the decreased level of [3 H] thymidine incorporation induced by VEGF-2 was tested. In contrast to the previous results, the addition of CNTF did not inhibit the proliferative response induced by VEGF (Figure 21C). These discoveries further substantiate that these two factors regulate the development of photoreceptor cells at different points in the lineage pathway.

DISCUSSION The above experiments identify and characterize the effect of VEGF-1 and VEGF-2 on retinal cells in vi tro. Treatment with VEGF in the subnanomolar range induces an increase in the number of photoreceptor and amacrine cells as well as increases in the level of rhodopsin protein and absorption of high affinity GABA. Temporal course studies demonstrate that VEGF induces a maximal increase in [3 H] thymidine incorporation within 48 hours of its addition and the delay of treatment of cultures for 24-48 h results in the loss of proliferative responses and of differentiation. The mitogenic response was regulated by the development with VEGF-2 inducing an increase in the incorporation of [3 H] thymidine with cells derived from animals E15, E20 and Pl. In comparison with the members of other families of trophic factors, the responses to treatment with VEGF-2 and FGF-2 were similar in that both factors increased the level of rhodopsin protein without resulted in the inhibition of the increase induced by VEGF at the rhodopsin level but not in the proliferative response. The VEGF receptor family is currently composed of four members (for a review see Klagsbrun and D'Amore, 1996; Wen et al., 1998). The receptors demonstrate a specificity for the overlapping ligand yet different. VEGFR-1 (Flt-1) and VEGFR-2 (Flk-2) bind to various forms of VEGF-1; whereas, VEGFR-2 and VEGFR-3 (Flt-4) bind to VEGF-2. In this way both of the VEGF-1 and VEGF-2 activate VEGFR-2 (Joukov et al, 998) and both ligands have similar biological activities in retinal cultures. Recently, Yank and Cepko (1996) described the developmental expression pattern of VEGF-2 in the retina. The degree of expected expression of the receptor on the newly formed vasculature, the receptors were also present on components of the neural retina. This pattern of expression was maintained during development while the retina grows centripetally. The effect of developmental age on the response of retinal progenitor cells to VEGF is consistent with the developmental pattern of receptor expression (Yang and Cepko, 1996). The mitogenic effects of VEGF, based on [3 H] thymidine incorporation studies, were noted at the initial developmental time point -? ai¿- examined, E15, as well as in E20 and Pl. The magnitude of the proliferative effect increased with age reaching a pibo in Pl. VEGF-2 is more effective in crops E15 and E20 than in Pl since the response was saturated at 1 as opposed to 10 ng / ml, respectively. In addition, the basal level of proliferation in vi tro also changed with developmental age with the highest levels observed in E17. The finding that the basal level of proliferation was relatively low in E15 but increased 4-fold with a 2-fold increase in cell density, a proportional increase greater than that observed at other developmental ages, suggests that endogenous mitogens may underlie the desensitization that occurs with treatment with VEGF-2 in E15 cultures. In addition, these data indicate that increased levels of VEGF during initial development may have a negative impact on the differentiation of photoreceptor cells. The influence of developmental age on the response of retinal progenitor cells to other growth factors has also been observed (Atshuler and Cepko, 1992). Lillien and Cepko (1992) reported that the proliferative response of retinal cells in monolayer FGF-1 cultures and FGF-2 was higher in initial stationary stages (for example E15 and E18) and by E21 or PO in the deviation to the right on the dose response curve was evident. . --- yes Previous studies in goldfish and frogs have suggested that the development of amacrine cells is regulated by cell-cell contact (Negishi et al., 1982; Reh and Tully, 1986). More recently, the importance of cell-cell contact for the development of photoreceptor cells in vi tro was also described by Wantabe and Raff (1990, 1992) in reaggregated cultures and later by Altshuler and Cepko (1992) with dissociated retinal cells cultured in gels. of collagen. In the first study, when E15 retinal cells were reaggregated with a 50-fold excess of neonatal retinal cells, there was no change in the time of development when rhodopsin-immunopositive cells were observed. However, there was a significant increase in the proportion of E15 cells that eventually differentiated into photoreceptor cells. In the case of the monolayer cultures used in this study, there was a dissociation between the initial or early proliferative response induced by VEGF-2 and delayed differentiation of photoreceptor cells. For example, VEGF-2 increases the incorporation of [3 H] thymidine 3-4 fold in cultures seeded at densities as low as 212 cells / mm 2 and treatment for 7 days resulted in cell densities equivalent to those reached at higher culture densities (for example 425 cells / mm2). However, there were no cells with rhodopsin protein or rhodopsin inmupositive ~ 7 detectable. These results suggest that not only is there a critical cell-cell interaction necessary for the development of photoreceptor cells but also a time frame during which the stimulus produced via cell contact is probably necessary The comparison of the time course of induced proliferation by VEGF with the time course of the development of the appearance of the protein rhodopsin indicates that there is a period of time of approximately 5 days between the two events. The appearance of the rhodopsin protein probably reflects the induction of genetic transcription since the two events have shown to be closely related (Treisman et al., 1988). This time interval is similar to that observed by Morrow et al (1998) in in vivo and in vi tro studies when progenitor cells derived from animals are considered within an age range of E20 to P3. In addition, between 5 and 9 days in vi tro, the greatest increase in the level of rhodopsin protein was observed and this period of time is within the period of postnatal development (day 6-10) in vivo during which there is a pronounced appearance of immunopositive cells to rhodopsin (Morrow et al., 1998). The correlation in these windows of time of development suggests that although VEGF-2 induces the proliferation of progenitor cells ^ |? I ^^^^^ ^^^^ ¿^ g "^^ j ^ photoreceptor, does not induce a significant delay in the differentiation of photoreceptor cells. As might be expected, if the progenitor cells were prevented from leaving the cell cycle. Compared to members of other trophic factor families, the response to VEGF-2 resembles that of FGF-2 in that both factors increase the level of rhodopsin protein while inducing relatively small increases in the total number of retinal cells. after 9 days in vi tro. In addition, a proliferative response, based on the incorporation of [3H] thymidine and cell counts, was noted to FGF-2 by Lillien and Cepko (1992) as late as P3 suggesting that FGF-2 retains some mitogenic activity in postnatal cultures. In contrast to our findings with VEGF-2, Fontaipe et al. (1998) showed that FGF-2 also has a survival effect on photoreceptor cells derived from P5 animals (data not shown). Treatment with TGFβ-1 resulted in a decrease in both the number of retinal cells and the level of rhodopsin protein. Kimichi et al. (1998) reported similar observations using human fetal retinal cultures with the exception that maximal inhibition with human cells required 0.5 ng / ml of TGFB-1 compared to less than 0.1 ng / ml required in rodent cultures.

, --- .. - .. ------ It is known that CNTF, a member of the neuropoyética family of cytokines, affects the development of photoreceptor cells in vitro and in vivo to increase the survival of photoreceptor cells after damage induced by light (Unoki and LaVail, 1994; Fuhram et al., 1995; Ezzeddine et al., 1997; Cayouette et al., 1998). In contrast to CNTF, VEGF-2 does not rescue photoreceptor cells in the damage model induced by constant light (La Vail et al., 1992; Wen et al., 1995; R. Wen and R. Alderson, unpublished data. ). Treatment of retinal explant cultures of postnatal rat with resulting CTF given as an increase in the number of cells expressing markers bipolar cell loss in the population of cells expressing rhodopsin. The analysis of the effect of CNTF on the fate of PO retinal cells labeled with [H] thymidine suggests that the cytokine does not induce the proliferation or increase in the survival of this cell population (Ezzeddine et al., 1997). In addition, the onset of the CNTF effect occurred at approximately the time when the cells became postmitotic and began to express rhodopsin. These data are consistent with the findings reported here showing that CNTF inhibits the increase induced by VEGF-2 in rhodopsin protein observed between 5 and 7 days in culture, but without its mitogenic activity observed between 1 and 2 days - -teaf--? .-j During the course of development in the retina oxygen levels control the microarchitecture of the vessels . / retinals that in turn equal the pattern of differentiation of retinal neurons (Chan-Ling et al., 1990, Phelps, 1990). Stone et al., (1995) has shown that in the retina, the microglia and astrocytes respond to hypoxia VEGF synthesize and secrete which in turn induces the formation of vessels. The studies reported here suggests: - the initial differentiation events regulated by VEGF not only involve the formation of vessels but tambiéin proliferation of progenitor cells photoreceptor. This can eventually result in the coordinated development of numerous types of cells in the retina.

Example 9: Increased Response of Endothelial Cells to Cotreatment with VEGF-2 and Antibody The antibodies generated by HGS have been shown to bind to VEGF-2 by ELISA assays, but are not thought to bind to sites involved in interactions with the receptor . The monoclonal 13D was mapped to an epitope on the N-terminal side of the molecule and 13A2 monoclonal was mapped to an epitope on the C-terminus (see Figure 24). The polyclonal antibody recognizes a number of the reduced form (fluorescent red), ie the stimulated proliferation i will produce a stronger signal and the inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells. The materials used for the LEC proliferation assay with blue alamar include: Blue Alamar (Biosource Cat # DAL1100); DMEM with 10% FBS + PennStr¡ep + Glutamine + 75 mg BBE + 45mg Heparin (Growth medium); DMEM with 10% FBS + PennStrep + Glutamine (Means of Starvation); DMEM with 0.5% FBS + PennStrep + Glutamine (sample dilution medium); Fluorescence reader CytoFluor; 96-well plates; and LEC cells, The alamar blue test was carried out as follows. For timing purposes it is best to plant the cells on the 96-well plates on a Wednesday, switch to starvation media on Thursday, inoculate the samples on Friday, incubate during the weekend and then add blue alamar and incubate and read on Monday . The LEC cells were seeded in growth medium at a density of 5000 cells / well of a 96-well plate and placed at 37 ° C overnight. After the nocturnal incubation of the LEC cells, the growth media were removed and placed again with starvation medium incubated for another 24 hours at 37 ° C. After the second 24 hours of incubation, the cells were inoculated with the appropriate dilutions of protein samples (prepared in DMEM + 0.5% FBS) in wells in triplicate. Once the cells had been inoculated with the samples the plate was placed again in the incubator at 37 ° C for three days. Three days later, 10 μl of blue alamar standard was added to each well and the plates were again placed in an incubator at 37 ° C for 4 hours. The plate was then read at an excitation between 530nm and a 590nm emission using the CytoFluor fluorescence reader. The direct output was recorded in relative fluorescence units. The basal level of activity was observed with the starvation medium only. This was compared with the observed output of positive control samples (VEGF-1 and / or bFGF) and dilutions of HGS protein. Three different antibody preparations made by HGHS (2 mouse monoclonal antibodies, 13A2, 13D6 and one polyclonal rabbit antibody) were evaluated for their ability to modulate the response of ECLs to activation mediated by VEGF-2. It was previously determined that a proliferative response of the ECL could be observed at a concentration of 1000 ng / ml of VEGF-2. Therefore, samples of VEGF-2 at a concentration of 1000 ng / ml in DMEM were premixed with one of the three anti-VEGF-2 antibodies different (10 μg / ml) and were used in the alamar blue assay system to determine the influence on the proliferation of LEC. The controls in the first experiment included VEGF-2 only (1000 ng / ml), bFGF (10 ng / ml, positive control), IL-2 (negative control of irrelevant protein) and starvation medium (negative control of the 1 J. . - , test) . The repeated experiment also included antibody alone (10 μg / ml) as a negative control. As shown in Figure 2, VEGF-2 treatment of ECLs at a concentration of 1000 ng / ml resulted in a proliferative response relative to negative controls, which was consistent with previous proliferative assays conducted with those cells . The simultaneous treatment of ECL with monoclonal VEGF-2 and i 13A2 did not increase the proliferative response above the level achieved with VEGF-2 alone. However, an increased proliferative response was observed cor. monoclonal 13D6 and to a lesser degree, with rabbit polyclonal antibody. As shown in Figure 23, the experiment was repeated under stricter conditions, using 1000 cells / well as an initial concentration and included stimulation with the antibody only to control possible direct effects of the antibodies on the ECL. This experiment showed increased proliferation mediated by VEGF-2 by 13D6 and polyclonal antibodies above the proliferative response observed with VEGF -2 or antibodies alone. As observed in the previous experiment, the 13A2 antibody did not induce an increased proliferative response.

These observations suggest that the cross-linking of VEGF-2 molecules bound to receptors (VEGFR2 or VEGFR3, mediated by the antibody can induce receptor dimerization.) This process can be used to enhance the signaling resulting from the binding of VEGF-2 to your receivers Example 10: Immunization of Mice for the Production of Monoclonal Antibodies The animals were housed individually and received food and water ad libi tum. All manipulations were performed using aseptic techniques. The experiments were conducted according to the rules and guidelines of Human Genome Sciences, the Committee on Care and Use of Institutional Animals and the Guidelines for the Care and Use of Laboratory Animals. Diluted protein concentration in 350 μl of phosphate buffered solution (PBS), or other neutral buffer, at a final protein concentration of 0.43 mg / ml With 0.35 ml of Freund's Complete Adjuvant, the adjuvant solution was emulsified and protein for a period of 10 minutes using two 3-ce glass syringes and a disposable three-way stopper (Baxter Cat. No 2C6240) To test the quality of the emulsion, they were i ~ t. . - »« - gsjl ..5 - afe »50 μls of the emulsion on the surface of the cold water in a beaker. If the emulsion does not remain as an intact white drop, then additional mixing is required. Extract all of the emulsion in a syringe, and use a 27-gauge needle, inject the mouse subcutaneously with a total of 200 μls of emulsion distributed between 4- sites, including the axillary and inguinal areas, the back of the neck, and along the back. After two or three weeks, repeat the previous injection replacing Freund's Incomplete Adjuvant (as opposed to Freund's Complete Adjuvant). After an additional two to three weeks, a third injection is given as discussed above, ensuring the use of Freund's Incomplete Adjuvant. Ten to fourteen days after the third injection, obtain 100-20 μls of mouse sagre per seeding tail vein. Incubate the blood at 37 ° C for 60 minutes, and then allow to cool overnight at 4 ° C. After incubation at 4 ° C, centrifuge the blood for 10 minutes. Transfer the serum to a new tube, and test the title of the mouse serum. If the titre is found to be low, intraperitoneal injections (i.p.) may be given at biweekly intervals. For i p injections, prepare 10-20 μg of protein per mouse in a volume of ---- 200-400 μls of PBS per mouse. Using a syringe! 1 ce and a 26-gauge needle, inject the solution into the mouse, Make a second bleed of the tail 10-14 days after the injection, and retest the serum of the mouse.

Example 11: Mouse Serum Title ELISA Coat the ELISA plate with 50 μl / well of purified antigen at 2 μg / ml PBS. Cover the ELISA plate with paraffin film and incubate at 4 ° C overnight in a humid chamber. After incubation, wash the plate 4 times with 200 μl / well of PBS per wash. Block with 3% BSA, 200 μls / well for 60 minutes at room temperature. Shake the blocking solution. Add serum samples in duplicate, 50 μls / well, at dilutions of 10"2, 10 ~ 3, 10" \ 10 ~ 5, 10"6 and 10" diluted in PBS with a content of 0.1% BSA. Include shock absorber blanks as well as positive and negative control serum at previous dilutions. Incubate at room temperature for 1-2 hours. Wash with PBST (PBS with 0.05% tween), 250 μls / well, four times. Add 50 μls / well of Biotinylated Anti-Mouse IgG at a concentration of 0.5 μg / ml in PBST, with a content of 0. 1% BSA and 2% horse serum. Incubate at temperature »*» Ti.j -. W - a ------ environment for 30 to 60 minutes. Wash the plate four times with PBST. Add 50 μls / well of ABC reagent (Vector Cat No, PK-6100) to the plate and incubate at room temperature for 30 minutes. Wash the plate six times with PBST Prepare the substrate for detection by ELISA by dissolving one tablet of tetramethylbenzidine dihydrochloride (TMB) (Sigma Cat. No. T-3405) 5 ml of ddH20. Add 5 more of Foisfato Citrate Buffer (25.7 ml of 0.2 M dibasic sodium phosphate, 24.3 ml of 0.1 M citric acid monohydrate, pH 5.0.). Add 2 μls of fresh 30% hydrogen peroxide, vortex vortex and use immediately, After incubation and washing the plate, add 100 μls of solution substrate and incubate at room temperature for approximately 15-30 minutes. Retain the reaction by adding 25 μls / well of 2 M H2SO4, and read the plate at 450 nm with 30 minutes against the controls.

Example 12: Fusion Protocol for the Production of Hybridoma One week before the fusion step, make the P3X growth medium (IX DMEM 0% (Gibco Cat. No. 11965-019) 5-10% fetal bovine serum, IX of L-Glutamine (Biofluids Cat.

No. 300) and IX of Sodium Piruvate (Biofluids Cat. No. 333). Thaw a new vial of P3X mouse myeloma cells in a well of a 6-well disk (see thawing protocol, infra) and start expanding in P3X growth medium. If the viability is good the next day transfer to a 100 mm disc. The cell density should not exceed 106 cells or more. In addition, the membranes of these cells should not look granular. In the díc. of fusion procedure there will be 6-8 plaques at 5-8 x 10b cells / ml. It is a good idea to try some P3X cells in HAT medium. All cells should die within approximately 4 days. If not then the P3X cells should be grown in P3X medium with a content of 15 μg / ml 8-azaguanine to eliminate revertants Four days before the fusion procedure, the mouse should be immunized with an injection of approximately 10 μg of high purity protein. One day before the fusion, divide the P3X 1 cells and feed them with fresh medium as needed, so that the cells remain healthy and grow in the logarithmic phase the next day. On the day of the fusion procedure, place 50 mis in P3X medium, PEG solution and HAT media (IX 0% DMEM, 20% fetal bovine serum, 4% HL Spiked with! BM Supplemented with Hybridoma (Boehringer-Mannheim), IX of L-Glutamine, IX of NEAA, IX of Pyruvate of Sodium, IX of HAT Sigma Cat. No. H0262), IX of 2ME 0.05M and IX of Penicil: ina-Streptomycin) in a water bath at 37 ° C . Approximately 100 ml of cold 0% DMEM are available. Check all P3X plates for possible contamination and to assess the health of the cells. Resuspend cells from 4 plates or flasks and combine 50 ml tubes. Centrifuge at 200 x G for 10 min. Aspirate the supernatant. Resuspend each tube with 10 ml of 0% DMEM and meeting. Count live cells using viability staining with trypan blue (viability should be greater than 90%). The total number of cells should be 2-4 x 107 cells. If there are not enough cells then repeat the process with a few more plates. Leave plates resting at room temperature (ART) until additional steps are needed. Prepare the bell where the spleen will be removed with: 70% EtOH, sterile instruments, including sieve and plunger, 2 petri dishes containing 10 ml of 0% DMEM and 15 ml of centrifuge tubes (2). The mouse is sacrificed, and the spleen is then removed from the skeleton. Place the spleen in the petri dish containing 10% DMEM. Place the sieve on another disk containing 10 ml DMEM 0% and cover with the lid of the box.

..., »Transfer the spleen using a pair of sterile forceps, and use the syringes with needles, put the spleen aside so that the cells are leaked to the media. Next, use the other plunger, gently squeeze the spleen through the sieve. Avoid crushing organ tissue through the sieve as this will result in strong fibroblast growth, remove the sieve and transfer the suspension of spleen cells to a 15 ml centrifuge tube. Wash the remaining cells of the disc with 5 ml of 0% DMEM, and add to the tube. Let the tube sit for 5 minutes to allow the large debris to settle to the bottom. Then transfer the cell suspension, minus the remains, to the second 15 ml tube. Centrifuge the cells for 10 min at 200 x G. Aspirate s / n and resuspend the spleen cells in 5 ml of 0% DMEM. Add 5 more of DMEM to 0%, and transfer the entire volume to the 50 ml tube. Remove 10 μls of the spleen cell suspension, and add 500 μls of trypan blue to count the lymphocytes. (Note: usually one spleen consistently produces 108 lymphocytes). ',, i »ato-t8 > itt-A .., - v. --t- - Fusion To the 50 ml centrifuge tube containing the spleen cells add enough P3X cells to produce a ratio of lymphocytes to P3X cells of 5: 1 (for example for 108 lymphocytes you will need 2 x 107 P3X cells) . Bring the total volume to 45-50 ml with 0% DMEM, and centrifuge at 200 x G for 10 minutes. Prepare a transfer hood with a stopwatch, hot PEG, hot P3X media, and a beaker of water at approximately 38-40 ° C. Aspirate all the supernatant of P3X lymphocyte sediment and try to loosen the sediment by hitting the tube. Place the tube in a small water bath. Keep the infusion tube in warm water, and shake gently, add 1 ml of PEG per drip for 1 minute. Then let stand with occasional shaking for 1-2 minutes, after which add 1 ml of P3X media per drip for one minute. Then add 3 mis of P3X media per drip for 1 minute, followed by the addition of 10 mis of P3X media per drip for 1 minute. I Gently add P3X media to make a total volume of 45 mis. Allow the tube to sit for 10 minutes, then centrifuge at 200 x G for 10 minutes. Aspirate the supernatant and gently resuspend the sediment in 5 or less than half HAT. Transfer the cell suspension to the bottle containing 400 ml of HAT medium and shake to mix. Pour some of the cell suspension into a sterile reservoir Place the cells in 96-well plates, 200 μls / well, using a 12-channel pipettor with filter tips. Place the plates in the incubator. Check plates for hybridoma growth or contamination. Leave the plates to incubate for 3 days. The first feed (change of medium) is carried out around day 7 by sucking approximately half of the media in each well using the 8-position manifold and replacing it with 100-150 μls / well of HT medium. Feed one week or even before the first separation to help remove any antibody produced by the unfused lymphocytic cells that has been found to continue producing antibody after two weeks in culture. Many or all wells will be ready to be shown by separation 2 weeks after the fusion when the colony or colonies fill more than half of the well and the supernatant has changed color to orange / yellow.

Example 13: Separation of Mouse Hybridomas by ELISA To separate mouse hybridomas, coat the ELISA plate (microtiter plate with U-shaped bottom Immulon 2 (Dynatech Cat. No. 011-010-3555)) with 50 μls / well of antigen at 2 μg / ml of PBS. Cover the ELISA plate with a plastic seal and incubate at 4 ° C overnight. After incubation, wash the plate 4 times with 200 μls / well of PBS per wash. Block with 2% BSA, 200 μls / well for 60 minutes at room temperature, shake the blocking solution. Add hybridoma supernatants, 150 μls / well and in 96-well assay plate, then transfer: go 50 μls of each supernatant from the assay plate, into the ELISA plate; include targets of culture media, as well as positive and negative mouse serum controls. Incubate at room temperature for 1-2 hours, or overnight at 4 ° C. Wash with PBST (PBS with 0.05% tween), 250 μls /. { Well, four times. Add 50 μls / well of Biotinylated Anti-Mouse IgG H + L, at a concentration of 0.5 μls / well in PBST with a content of 0.1-0.3% BSA and 1% horse serum. Incubate at room temperature for 30 to 60 minutes. Wash the plate four times with PBST. Add 50 μls / well of a reagent (Cat. No. PK-6100) ABC to the plate and incubate at room temperature for 30 minutes. Wash the plate six times with PBST.

Prepare the substrate for detection by ELISA by dissolving one tablet of tetramethylbenzidine dihydrochloride (TMB) (Sigma Cat. No. T-3405) in 5 mls of ddH20. Add 5 mis of Phosphate Buffer Citrate 0.1M (25.7 mis of dibasic sodium phosphate 0.2 M, 24.3 ml of 0.1 m citric acid monohydrate, pH 5.0). Add 2 μls of 30% fresh hydrogen peroxide, vortex vortex and use immediately, After incubation and washing the plate, add 100 μls of substrate solution and incubate at room temperature for approximately 15- 30 minutes. Stop the reaction by adding 25 μls / well of 2M H2SO4 and read the plate at 450 nm for 30 minutes against the controls.

Example 14: Proof of Relative Affinity of Monoclonal Antibodies Derived from Culture Supernatants A. Determination of Antigenic Coating Concentration Make approximately 1 ml of the antigen at a concentration of 4 ug / ml in PBS. Transfer to a microdilution tube. Place 0.5 ml of PBS in each of 9 microdilution tubes, then serial dilutions. from 1/2 transferring 0.5 ml of tube to tube starting from 4 ug / ml of tube. Now you will have tubes containing 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015 and 0.0075 ug / ml. Recap a plate with the previous concentrations, 6 wells each, 50 ul / well. Cover or incubate overnight at 4 ° C. After incubation, wash the plate four times with 200 μls / well of PBS per wash. Block with 3% BSA, 200 μls / well for 60 minutes at room temperature. Shake the blocking solution. . Observe in the curve of the serum title of mouse which is positive to the antigen. Determine the dilution of the serum that is just above the titration curve Add the positive mouse serum to this dilution in PBS with a content of 1% BSA, 50 μls / well, rows B-D, columns 2-11. Include negative control serum at the previous dilution in the E-G rows, columns 2-11. Incubate overnight at 4 ° C. After incubation, subtract the Negative Control Serum values from the Positive Control Serum values. Graph the average value of (O.D. 450) against the antigen concentration on a linear scale. Determine the coating concentration of the antigen that O.D. submaximal. This is the concentration of the coating to be used for the relative affinity test.

B. Determine the concentration of mouse IgG from the hybridoma supernatant sample using the "Mouse IgG ELISA" Kit from Boehringer Mannheim Biochemistry (Cat. No. 1333 151) Dilute the 1/10 coating buffer concentrate with ddH20. Twenty more will be necessary of di, e, z. Obtain an aliquot of the capture antibody. Thaw 3 tubes of Concentrated Shock Absorber (blocking solution). Twelve wells by standards and 4-6 wells will be needed for each supernatant to be tested. Calculate the number of mLs of diluted Capture Antibody needed to assume 50 μls / well of coating volume. Dilute the Capture Antibody in the following ratio: 25 ul Capture Ac = X ul Capture Ac 1 ml Shock Absorber # ml Shock Absorber Coating Coat the Nunc plate with the solution and incubate for 30 minutes at room temperature on a shaker. Dilute the Shock Absorber Concentrate 1/10 in ddH20. Wash the plate in the wash buffer with ELISA (0.9% NaCl, 0.1% Tweeen 20) and block with 200 uls / well of Post-Recovery Shock absorber (Blocking solution) for 15 minutes at room temperature. Dilute the standard IgG in Post-Recovery Shock Absorber (blocking solution) in the following concentrations: 0.2, 0.1, 0.05, 0.025, 0.0125 and 0 00625 ug / ml in Post-Coating Shock Absorber.

C. Relative Affinity Test Coat the appropriate ELISA plates overnight at 4 ° C with the previously determined antigen concentration. Block the plate as before. Make 1/3 serial dilutions in PBS + 0.1% BSA from the test supernatant Add 50 uls / well of the dilutions of the supernatant sample, including the undiluted sample, to the ELISA plates in duplicate or triplicate. The positive control consists of a few wells of positive control mouse serum at the same concentration used to determine the antigen coating concentration. The negative control consists of a few wells of the dilution buffer. Cover and incubate overnight at 4 ° C. Graph the IgG concentration of each supernatant against the average value of (O.D. 450) in a 4 parameter curve fit. The curves of the supernatant that are further to the left are the supernatants with the highest affinities.

Example 15: Production of Ascitos in Mice Hybridoma cells should be healthy and in logarithmic growth phase for the production of ascites. Transfer the cells to a 15 ml tube and coritar.

Determine the volume containing 4 x 10 cells, transfer that volume to a second tube and centrifuge the cells. Resuspend the sediment in 0.9 ml of HBSS ^ Hank's Balanced Saline Solution) and transfer to an eppendorf tube. Fill 1 syringe of ce with the cell suspension and inject the ip mice as follows: 0.2 ce per mouse if the original cell number was 4xl06 and 0.3 ce if the original cell number was 3xl06. When the abdomen is very distended and slightly bulging to the touch, such as a balloon, (usually on day 9 or 10 but sometimes later up to 14 days) then it is time to "drain the mouse A. Drained: Hold the mouse in your left hand and use an alcohol swab to clean the abdomen area just above the left rear leg of the mouse. While holding the mouse over an open 15 ml centrifuge tube, insert a 19 gauge needle into the abdomen. The ascitic fluid should start immediately falling from the end of the needle into the centrifuge tube. An average mouse should produce 3-6 ml of ascitic fluid.

B. Processing and Storage of Ascitps: Collect the ascitic fluid collected from each mouse in the group (all injected with the same hybridoma) and leave at room temperature for 1-2 hours and place at 37 ° C for 15-30 minutes. Then place the asci ations 4 ° C at night to allow the formation of a clot. Centrifuge the coagulated shoots for 10 minutes. Transfer the liquid ascitos to a 50 ml centrifuge tube, and store the tube at -20 ° C. Afterwards the drains can be added to this 50 ml tube. When all mice are killed, the assembled ascitos can be thawed, recentrifuged, and placed in aliquots for long-term storage at -20 ° C or -70 ° C. The ascitos will have to be titled by ELISA: Example 16: Protocol for Freezing and Thawing Hybridoma and Mouse Myeloma Cells A. Freezing The cells to be frozen should be healthy, in logarithmic growth phase of a concentration of approximately 5 x 10 5 cells / ml. Resuspend cells from a 6 well plate or flask, transfer to a 15 ml tube and count. Calculate the number of total cells and divide by ^^^ Lk ^ v &fj ^^^^? J * ^ ¡i 1-3 x 106 cells per bottle to determine the number of bottles to be frozen. Sediment the cells at 200-300 G for 5-10 minutes. Aspirate the supernatant from the pellet and resuspend in sufficient freezing medium (50% FBS, 10% DMSO in DMEM, or Original DMSO Freezing Medium (IGEN), Fisher Cat. No. IG-50-0715) to achieve the number desired cell / flask per ml (cell densities should be in the range of 5 x 10 5 to 1 x 10 7 cells). Immediately transfer the cell solution to criofrascos, 1 ml per bottle, and place on ice. Transfer the flasks to a controlled speed freezer and place the freezer at -70 ° C overnight. 24 hours later transfer the bottles to a liquid nitrogen tank or a freezer at -130 ° C for long-term storage.

B. Thawing Add 10 ml of cold media (eg P3X media) to a 15 ml tube. Recover the frozen cell cryophone and keep on dry ice until thawing begins. Quickly thaw the cells in a 37 ° C bath. Hold the bottle during defrosting and keep stirring gently until there is only a small piece of ice in the bottle. Do not allow content to be > »T-ai? -, - fo • ---- # * $ * •. • - ^. YA ^ AB hot above 4 ° C. Keep the alcohol and t out of the crayfish. Use a sterile pasteur pipette and without touching the edges of the cryoprobe, transfer the cell suspension in the bottle to the 10 ml of cold media. Centrifuge at 200-300 G for 5-10 minutes. Aspirate the supernatant and resuspend the pellet in 6 ml of Cloning Media HT (supra). Transfer to 1 well of a 6-well disc. Evaluate viability 24 hours later. The viability must not be less than 50% Example 27; In Vitro Assay for the Angi Protein Activity The following assay is designed to detect the activity of the angiogenic protein, preferably the activity of VEGF-2. For example, a chimeric receptor is generated by fusing the nucleotides encoding the extracellular domain of the Flt-4 receptor (SEQ ID NO: 27) (Galland et al., Genomics 13 (2): 475-478 (1992), which are hereby incorporated by reference in their entirety) to the nucleotides encoding the transmembrane domain and the intracellular domain of Flk-1 (SEQ ID. NO: 28) (Davis-Smith et al., EMBO J 15 (18) 4919-4927 (1996), which are incorporated herein by reference in their entirety) thus, the chimeric receptor would include amino acids 1 to 775 of SEQ ID NO: 27, fused to amino acids 765 to 1356 of SEQ ID NO: 28, respectively. Alternatively, the chimeric receptor may be designated as set forth above, but it would replace the transmembrane and intracellular domains of the erythropoietin receptor (EPOR) with the transmembrane and intracellular domains of the Flk-1 receptor, as described in Pacifici et al. , JBC 269 (3): 1571-1574 (1994), which is incorporated herein by reference in its entirety (see especially Figure 1). The resulting DNA encoding the chimeric receptor is cloned into an appropriate mammalian, baculoviral or bacterial expression vector, such as, for example, pC4, pCDNA3, or pA2, as discussed supra. Mammalian host cells that could be used for the expression of the chimeric receptor include NIH3T3 (supra), or the BaF3 pre-B cell line (Achen et al., PNAS 95 (2): 548-553 (1998), which is incorporated herein by reference in its entirety.To test the activity, the angiogenic protein can be contacted with a cell line that expresses the chimeric receptor, or extracts thereof, then the angiogenic protein that binds to the protein. The chimeric receptor can be detected by measuring any resulting signal transduced by the chimeric receptor Numerous modifications and variations to the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention can be practiced in another way to the particularly described All the description of all publications, ^ including patents, patent applications, articles of work, manuals of laboratory, books or other documents cited herein are incorporated by reference Additionally, the entire specification, including the Sequence Listing of the US Application No.

Series 09 / 107,997, filed on June 30, 1998, and PCT Application Number US 99/05021 filed on March 10, 1999 is incorporated herein by reference in its entirety. 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 SEQUENCE LIST < 310 > Human Genome Sciences, Inc. et al. < 120 > Factor 2 of Vascular Endothelial Growth < 130 > PF112 PCT4 < 140 > PCT / USOO / 03047 < 141 > 2000-02-07 < 150 > 60 / 119,179 < 151 > 1999-02-08 < 150 > 60 / 119,926 < 151 > 1999-02-12 < 150 > 60 / 137,796 < 151 > 1999-06-03 < 150 > 60 / 171,505 < 151 > 1999-12-22 < 160 > 37 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 1674 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > < 12) .. (1268) < 220 > < 221 > sig_peptide < 222 > (12) .. (80) < 220 > < 221 > mat_peptide < 222 > (811) .. (1268) < 400 > 1 gtccttccac c atg falls tcg ctg ggc ttc ttc tet gtg gcg tgt tet ctg 50 Met His Ser Leu Gly Phe Phe Ser Val Wing Cys Ser Leu -20 -15 etc gcc gcg ctg etc cgc ggt cct cgc gac gcg ccc gcc gcc gcc 98 Leu Wing Wing Wing Leu Pro Gly Pro Arg Glu Wing Pro Wing Wing Ala -10 -5 -1 1 5 gcc gcc ttc gag tec gga etc gac ccc tcg gac gcg gag ccc gac gcg 146 Wing Wing Phe Glu Ser Glu Leu Asp Leu Ser Asp Wing Glu Pro Asp Wing 10 15 20 ggc gag gcc acg gct tat gca age aaa gac 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 tet gtg tec agt gta gat gaa etc atg act gta etc tac cea 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 etc aac tca agg here gaa gag act ata aaa ttt 338 Arg Glu Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr He Lys Phe 75 80 85 gct gca gca cat tat aat here gag atc ttg aaa agt att gat aat gag 386 Ala Ala Ala His Tyr Asn Thr Glu He Leu Lys Ser He Asp Asn Glu 90 95 100 tgg aga aga tga ac tgc atg cea cgg gag gtg tgt ata gat gtg ggg 434 T rp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Cys He Asp Val Gly 105 110 115 aag gag ttt gga gtc gcg here aac ac ttc ttt aaa cct cea tgt gtg 482 Lys Glu Phe Gly Val Wing Thr Asn Thr Phe Phe Lys Pro Pro Cys Val 120 125 130 tec gtc tac aga tgt ggg 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 ac age age tac etc age aag acg tta ttt gaa att here 578 Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu He Thr 155 160 165 gtg cct etc tet ca ggc ccc aaa cea gta here atc agt ttt gcc aat 626 Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr He Ser Phe Wing Asn 170 175 180 falls act tec tgc cga tgc atg tet aaa ctg gat gtt tac aga ca gtt 674 His Thr Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val 185 190 195 cat tec att att aga ctgt tec ctg cea gca here cta cea cag tgt cag 722 His Ser He Arg Arg Seu Leu Pro Wing Thr Leu Pro Gln Cys Gln 200 205 210 gca gcg aac aac acc tgc ccc acc aat tac atg tgg aat aat cae 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 tg ttt tec tcg gat gct gga 818 Cys Arg Cys Leu Wing Gln Glu Asp Phe Met Phe Be Ser Asp Gly 235 240 245 gat gac tca here gat gga ttc cat gac atc tgt gga cea 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 tgc gtc tgc aga gcg ggg ctt cgg cct 914 Leu Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro 265 270 275 gcc age tgt gga ccc falls aaa gaa cta gac aga aac tca tgc cag tgt 962 Wing Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys 280 285 290 gtc tgt aaa aac aaa etc tcc ccc age cag tgt ggg gcc aac cga gaa 1010 Val Cys Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu 295 300 305 310 ttt gat gaa aac here 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 ca ccc cta aat cct gga aaa tgt gcc tgt gaa tgt here gaa agt 1106 Asn Gln Pro Leu Asn Pro Gly Lys Cys Wing Cys Glu Cys Thr Glu Ser 330 335 340 cea cag aaa tgc ttg tta aaa gga aag aag ttc falls falls here tgc 1154 Pro Gln Lys Cys Leu Law Lys Gly Lys Lys Phe His His Gln Thr Cys 345 350 355 age tgt tac aga cgg cea tgt acg aac cgc cag aag gct tgt gag cea 1202 Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro 360 365 370 gga ttt tca tat agt gaa 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 cea caa atg age taagattgta ctgttttcca gttcatcgat 1298 Gln Arg Pro Gln Met Ser 395 tttctattat ggaaaactgt gttgccacag tagaactgtc tgtgaacaga gagacccttg 1358 tgggtccatg ctaacaaaga caaaagtctg tctttcctga accatgtgga taactttaca 1418 ^^^^^^^ < 222 > (71 '1120) < 220 > < 221 > sig-peptide < 222 > (71) .. (142) < 220 > < 221 > mat peptide < 222 > (147) .. (1120) < 400 > 3 cgaggccacg gcccatgcaa gcaaagatct ggaggagcag ctacggtctg tgtccagtgt 60 agatgaactc atg act gta etc tac cea gaa tat tgg aaa atg tac aag 109 Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Net Tyr Lys -20 -15 tgt cag cta agg aaa gga ggc tgg ca 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 1 5 etc aac tca agg here gaa gag act ata aaa ttt gct gca gca cat tat 205 Leu Asn Be Arg Thr Glu Glu Thr He Lys Phe Wing Wing His Wing Tyr 10 15 20 aat here gag atc ttg aaa agt att gat aat gag tga aga aga act a 253 Asn Thr Glu He Leu Lys Ser He Asp Asn Glu Trp Arg Lys Thr Gln 25 30 35 tgc atg cea cgg gag gtg tgt ata gat gtg ggg aag gag ttt gga gtc 301 ^ $ ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡ 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 age acg 397 Gly Gly Cys Cys Asn Ser Glu GLy Leu Gln Cys Met Asn Thr Ser Thr 70 75 80 85 age tac etc age aag acg tta ttt gaa att here gtg cct etc tet caa 445 Ser Tyr Leu Ser Lys Thr Leu Phe Glu He Thr Val Pro Leu Ser Gln 90 95 100 ggc ccc aaa cea gta here atc agt ttt gcc aat falls act tec tgc cga 493 Gly Pro Lys Pro Val Thr He Ser Phe Wing Asn His Thr Ser Cys Arg 105 110 115 tgc atg tet aaa ctg gat gtt tac aga ca gtt cat tec att att aga 541 Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser He He Arg 120 125 130 cgt tec ctg cea gca aca cta cea cag tgt cag gca gcg aac aac acc 589 Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr 135 140 145 tgc ccc acc atat tg tgg aat aat 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 tec tcg gat gct gga gat gac tca here gat 685 .-ai- -. Vx »3b uÁ? 3k & Gln Glu Asp Phe Met Phe Be Ser Asp Wing Gly Asp Asp Ser Thr Asp 170 175 180 gga ttc cat gac atc tgt gga cea 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 age tgt gga ccc 781 Cys Gln Cys Val Cys Arg Wing Gly Leu Arg Pro Wing Cys Gly Pro 200 205 210 falls 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 etc tcc ccc age caà tgt ggg gcc acc cga gaa ttt gat gaa aac here 877 Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr 230 235 240 245 tgc cag tgt gta tgt aaa aga acc tgc ccc aga aat ca c c cta cta cta cta 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 here gaa agt cea cag aaa tgc ttg 973 Pro Gly Lys Cys Wing Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu 265 270 275 tta aaa gga aag aag ttc falls falls here tgc ag e tgt tac aga cgg 1021 Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg 280 285 290 cea tgt acg aac cgc cag aag gct tgt gag cea gga ttt tca tat agt 1069 Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser 295 300 305 gaa gaa gtg tgt cgt tgt gto cct tca tat tgg ca aga cea caa atg 1117 Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Gln Arg Pro Gln Met 310 315 320 315 age taagattgta ctgttttcca gttcatcgat tttctattat ggaaaactgt 1170 Gttgccacag be tagaactgtc tgtgaacaga gagacccttg tgggtccatg ctaacaaaga 1230 tctttcctga caaaagtctg accatgtgga taactttaca gaaatggact ggagctcatc tgcaaaaggc 1290 ctcttgtaaa gactggtttt ctgccaatga ccaaacagcc aagattttcc 1350 tcttgtgatt tctttaaaag aatgactata taatttattt ccactaaaaa tattgtttct 1410 gcattcattt ttatagcaac aacaattggt aaaactcact gtgatcaata tttttatatc 1470 atgcaaaata tgtttaaaat aaaatgaaaa ttgtatttat aaaaaa aaaaaaaaaa 1526 < 210 > 4 < 211 > 350 < 212 > PRT < 213 > Homo sapiens < 400 > 4 .- ^ AJiiSte 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 T [hr 140 145 150 sn Tyr Met Trp Asn Asn His He Cys Arg Cys Leu Wing Gln Glu Asp 155 160 165 Thr Asp Ser Arg Cys Lys Wing Arg Gln Leu Glu Leu Asn Glu Arg Thr 210 215 220 Cys Arg Cys Asp Lys Pro Arg Arg 225 230 < 210 > 8 < 211 > 14 < 212 > PRT < 213 > Homo sapiens < 220 > < 221 > SITE < 222 > (2) < 223 > Xaa equal to any amino acid < 220 > < 221 > SITE < 222 > (5) < 223 > Xaa equal to any amino acid < 220 > < 221 > SITE < 222 > (7) < 223 > Xaa equal to any amino acid < 220 > < 221 > SITE < 222 > (10) < 223 > Xaa equal to any amino acid < 400 > 8 Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn 1 5 10 < 210 > 9 < 211 > 18 < 212 > DNA < 213 > Homo sapiens < 400 > 9 atgctccgg ctcgtatg 18 < 210 > 10 < 211 > 112 < 212 > DNA < 213 > Homo sapiens < 400 > 10 aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg gataagaatt aagatgtacc 60 caattgtgag cggataacaa ttteacacat taaagaggag aaattacata tg 12 < 210 > 11 < 211 > 18 < 212 > DNA < 213 > Homo sapiens < 400 > 11 atgcttccgg ctcgtatg 18 < 210 > 12 < 211 > 19 < 212 > DNA < 213 > Homo sapiens < 400 > 12 gggttttccc agtcacgac 19 < 210 > 13 < 211 > 21 < 212 > DNA < 213 > Homo sapiens < 400 > 13 ccacatggtt caggaaagac to 21 h. ¿ííáe * < 210 > 14 < 211 > 50 < 212 > DNA < 213 > Homo sapiens < 400 > 14 tgtaatacga ctcactatag ggatcccgcc atggaggcca cggcttatgc 50 < 210 > 15 < 211 > 28 < 212 > DNA < 213 > Homo sapiens < 400 > 15 gatetetaga ttagetcatt tgtggtct 28 < 210 > 16 < 211 > 27 < 212 > DNA < 213 > Homo sapiens < 400 > 16 cgcggatcca tgactgtact ctaccca 27 < 210 > 17 < 213 > Homo sapiens < 400 > 20 gcagcacata tgacagaaga gactataaaa 30 < 210 > 21 < 211 > 30 < 212 > DNA < 213 > Homo sapiens < 400 > 21 gcatgcaggta cctcaacgtc taataatgga 30 < 210 > 22 < 211 > 30 < 212 > DNA < 213 > Homo sapiens < 400 > 22 gcagcaggat cccacagaag agactataaa 30 < 210 > 23 < 211 > 30 < 212 > DNA < 213 > Homo sapiens < 210 > 27 < 211 > 39 < 212 > DNA < 213 > Homo sapiens < 400 > 27 gcagggtacg gatcctagat tagctcattt gtggtcttt 39 < 210 > 28 < 211 > 38 < 212 > DNA < 213 > Homo sapiens < 400 > 28 gcagcaggat ccacagaaga gactataaaa tttgctgc 38 < 210 > 29 < 211 > 37 < 212 > DNA < 213 > Homo sapiens < 400 > 29 cgtcgttcta gatcacagtt tagacatgca tcggcag 37 . »* - * -_---. - -, «-. - - • "*" * < 211 > 35 < 212 > DNA < 213 > Homo sapiens < 400 > 33 gactggtacc ttatcacata aaatcttcct gagcc 35 < 210 > 34 < 211 > 39 < 212 > DNA < 213 > Homo sapiens < 400 > 34 gactggatcc gccaccatgc actcgctggg cttcttctc 39 < 210 > 35 < 211 > 34 < 212 > DNA < 213 > Homo sapiens < 400 > 35 gactggtacc ttatcagtct agttctttgt gggg 34 < 210 > 36 < 211 > 39 < 212 > DNA < 213 > Homo sapiens < 400 > 36 gactggatcc gccaccatgc actcgctggg cttcttctc 39 < 210 > 37 < 211 > 37 < 212 > DNA < 213 > Homo sapiens < 400 > 37 gactggtacc tcattactgt ggactttctg tacatte 37

Claims (41)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. A method for treating photoreceptor damage or degeneration, characterized in that it comprises administering to a subject suffering from such damage or degeneration of the photoreceptors a therapeutically effective amount of a vascular endothelial growth factor 2 (VEGF-2).
2. 2. The method of compliance with the claim 1, characterized in that the damage or degeneration of the photoreceptors is associated with angioid events, retinitis pigmentosa, Kearn syndrome, dystrophies of the pigmentation pattern, retinal perforations, retinitis, chorioretinitis, cytomegalovirus retinitis, acute retinal necrosis syndrome, alveolar choroidal dystrophy central, dominant drusen, hereditary hemorrhagic macular dystrophy, macular dystrophy of North Carolina, pericentral choroidal dystrophy, adult foveomacular dystrophy, benign concentric ring macular dystrophy, central aureolar pigment epithelial dystrophy, congenital macular coloboma, dominant hereditary macular edema, foveal retinoesquistosis family syndrome, bright fenestrated macular dystrophy, progressive foveal dystrophy, slowly progressive macular dystrophy, pseudoinflammatory Sorsby dystrophy, dystrophy of the ? ü - n - '. «s, - < .- .. 4L0 cones-rods, progressive cones dystrophy, congenital Leber amaurosis, Goldman-Favre syndrome, Bardet-Biedl syndrome, Bassen-Kornzweig syndrome (abetalipoproteinemia), Best's disease (viteliform dystrophy), choroidemia, spin atrophy, congenital amaurosis, Refsum syndrome, Stargardt's disease and Usher syndrome, age-related macular degeneration, diabetic retinopathy, peripheral vitreoretinopathies, photonic retinopathies, surgery-induced retinopathies, viral retinopathies, ischemic retinopathies, retinal detachment and traumatic retinopathy.
3. 3. The method according to claim 1, characterized in that the VEGF-2 comprises the amino acid sequence set out in SEQ ID NO: 4 or a variant, or a derivative thereof.
4. 4. The method according to claim 3, characterized in that the VEGF-2 comprises the amino acid sequence set forth in SEQ ID NO: 4.
5. 5. The method according to claim 1, characterized in that the VEGF-2 comprises the VEGF-2 linked to a water soluble polymer.
6. 6. The method according to claim 5, characterized in that the water-soluble polymer is polyethylene glycol.
7. The method according to claim 1, characterized in that the VEGF-2. it comprises a tricked VEGF-2.
8. The method according to claim 1, characterized in that the VEGF-2 is administered at a dose of between about 0.005 mg / kg and about 50 mg / kg of body weight.
9. 9. The method according to claim 8, characterized in that the VEGF-2 is administered at a dose between about 0.05 mg / kg and about 5 mg / kg of body weight.
10. 10. The method of compliance with the claim 1, characterized in that VEGF-2 is administered as a sustained release pharmaceutical composition.
11. The method according to claim 1, characterized in that the VEGF-2 is administered as a topical, oral or parenteral pharmaceutical composition.
12. The method according to claim 1, characterized in that the VEGF-2 is administered by cell therapy or genetic therapy means, where the cells have been modified to produce and secrete VEGF-2. .-1- .. -t .. -
13. 13. The method according to claim 12, characterized in that the cells have been modified ex vivo.
14. 14. The method according to claim 1, characterized in that the cells have been modified in vivo.
15. The method according to claim 1, characterized in that it further comprises administering to the patient an effective amount of a second therapeutic agent to treat retinal diseases
16. 16. The method according to claim 15, characterized in that the second therapeutic agent is selected from the group consisting of neurotrophic factor derived from glial cell lines, neurotrophic factor derived from the brain, neurotrophin 3, neurotrophin 4/5, neurotrophin 6, insulin-like growth factor, ciliary neurotrophic factor, growth factors of the acidic and basic fibroblasts, fibroblast growth factor 5, transformation growth factor, and the transcript regulated by cocaine-amphetamine, epidermal growth factor, inhibitory factor of leukemia, an interleukin, an interferon and a stimulating factor.
17. 17. The method according to claim 1, characterized in that the VEGF is administered by means of distribution and release selected from the medium consisting of ocular inserts, ocular injection or ocular implants.
18. 18. A method for providing photoreceptor cells for implants, characterized in that it comprises culturing the dissociated photoreceptor cells in the presence of VEGF-2.
19. 19. The composition characterized in that it comprises an isolated antibody, wherein the antibody binds especially to the polypeptide of SEQ ID NO: 2 or the polypeptide encoded by the human cDNA in a Deposit No. 75698 or 97149.
20. The composition according to claim 19, characterized in that it is selected from the group consisting of: polyclonal, monoclonal, chimeric, humanized, human antibody, a single chain antibody, antigen binding fragment, a murine antibody fragment, a human antibody fragment or a fragment of a single chain antibody.
21. The composition according to claim 20, characterized in that the single chain antibody fragment is selected from the group consisting of: an Fv fragment, a Fab fragment, a Fab 'fragment, a fragment of F ( ab ') 2, a light chain variable domain, a heavy chain variable domain, a ^ g faith ^ portion of the hinge region, a portion of the CH1 domain, a portion of the CH2 domain, a portion of the CH3 domain
22. 22 The composition according to claim 19, characterized in that the antibody binds specifically to a polypeptide that consists of residual amino acids selected from the group consisting of: amino acids -23 to +1 of SEQ ID N0: 1, residual amino acids 1 to 50 of SEQ ID NO: 2, residual amino acids 50 to 100 of SEQ ID NO: 2, residual amino acids 100 to 150 of SEQ ID NO: 2, residual amino acids 150 to 200 of SEQ ID NO: 2, residual amino acids 200 to 250 of SEQ ID NO: 2, residual amino acids 250 at 300 of SEQ ID NO: 2, residual amino acids 300 to 350 of SEQ ID NO: 2, residual amino acids 350 to 396 of SEQ ID NO: 2.
23. 23. The composition according to claim 19, characterized in that the antibody has a Kd selected from the group consisting of less than 1X10-10, less than 1X10-11, less than 1X10-12, less than 1X10-13, or less of 1X10-14.
24. 24. The composition according to claim 19, characterized in that the antibody binds only to the polypeptides encoded by the polynucleotides that hybridize to the polynucleotide sequence SEQ ID NO: 1 under stringent hybridization conditions.
25. 25. The composition according to claim 19, characterized in that the antibody does not bind to any other analogous, orthologous or homologous polypeptide sequence of SEQ ID NO: 2 or the full-length polypeptide encoded by the cDNA contained in the ATCC deposit No. 75698 or 97149.
26. 26. The composition according to claim 19, characterized in that the antibody does not bind to a polypeptide selected from the group consisting of: less than 95% identity of SEQ ID NO: 2 or a polypeptide of length complete encoded by the human cDNA contained in ATCC Deposit No. 75698 or 97149, the identity of less than 90% of SEQ ID NO: 2 or a full length polypeptide encoded by the human cDNA contained e :? ATCC Deposit No. 75698 or 97149, identity less than 85% of SEQ ID NO: 2 or a full-length polypeptide encoded by the human cDNA contained in ATCC Deposit No. 75698 or 97149, identity less than 80% of the SEQ ID NO: 2 or a full-length polypeptide encoded by the human cDNA contained in ATCC Deposit No. 75698 or 97149.
27. 27. The composition according to claim 19, characterized in that the antibody is selected from the group consisting of antibodies. monoclonal 12E2; 13A2; 15C2; 13D6; 13E6; 19A3; 8G11; 11A8, 15E10, 9B4 and 13G11. 8 & i
28. 28. A composition characterized in that it comprises: (a) a polypeptide fragment of SEQ ID NO: 2 or a polypeptide fragment encoded by the human cDNA contained in the ATCC NO. 75698 or 97149; and (b) an isolated antibody.
29. 29. The compliance composition as claimed in claim 28, characterized in that the antibody binds especially to the polypeptide of SEQ ID NO: 2 b the polypeptide encoded by the cDNA in the ATCC deposit No. 75698 or 97149.
30. 30. The composition according to with claim 28, characterized in that the antibody is selected from the group consisting of monoclonal antibody 12E2; 13A2; 15C2; 13D6; 13E6; 19A3; 8G11; 11A8, 15E10, 9B4 and 13G11.
31. The composition according to claim 28, characterized in that the polypeptide fragment contains a fragment selected from the group consisting of: an N-terminal deletion fragment described by the general formula m-396 of SEQ ID NO: 2, a C-terminal deletion fragment described by the general formula -23-n, an N-terminal and C-terminal deletion fragment described by the general formula mn, a C-terminal deletion fragment described by the + 9-n, a mature fragment of SEQ ID NO: 2 or encoded by the human cDNA in ----- * -. 4L7 ATCC Deposit No. 75698 or 97149, a proprotein fragment of SEQ ID NO: 2 or encoded by the human cDNA in ATCC Deposit No. 75698 or 97149, or a full length fragment of SEQ ID NO: 2 or encoded by the. human cDNA in ATCC Deposit No. 75698 or 97149.
32. 32. The composition according to claim 28, wherein the polypeptide fragment comprises amino acid residues R-204 to S-396 of SEQ ID NO: 2 or amino acid residues F-9 to R-204 of SEQ ID NO: 2.
33. 33 The composition according to claim 28, wherein the composition comprises a first polypeptide fragment of amino acid residues R-204 to S-396 of SEQ ID NO: 2 and a second polypeptide fragment of amino acid residues F-9 to R -203 of SEQ ID NO: 2.
34. 34. A composition characterized in that it comprises: (a) a first polypeptide fragment of SEQ ID NO: 2 or a first polypeptide fragment decoded by a human cDNA contained in the ATCC deposit No. 75698 or 97149; and (b) a second polypeptide fragment of SEQ ID NO: 2 or a second polypeptide fragment encoded by the human cDNA contained in the ATCC deposit No. 75698 or 97149; é / - aÉ-fc-. wherein the first polypeptide fragment is linked to the second polypeptide fragment.
35. 35. The composition according to claim 34, characterized in that the binding is effected by a selected member consisting of: an antibody, a hinge, a glycine binder, a serine binder or a proline binder, non-covalent interactions, disulfide bonds or covalent interactions.
36. 36. A method of endothelial cell proliferation in a patient, characterized in that it comprises administering to the patient the composition according to claim 19.
37. 37. A method of proliferating endothelial cells in a patient, characterized in that it comprises administering to the patient the composition of according to claim 2
38. 38. A method of proliferating endothelial cells in a patient, characterized in that it comprises administering to the patient the composition according to claim 34.
39. 39. The method according to claim 36, characterized in that the method stimulates the angiogenesis or lymphangiogenesis
40. 40. The method in accordance with the claim 37, characterized in that the method stimulates angiogenesis or lymphangiogenesis.
41. 41. The method according to the claim 38, characterized in that the method stimulates angiogenesis or lymphangiogenesis.