MXPA97009570A - Growth factor 3 endothelial, vascular, hum - Google Patents

Abstract

Polypeptides of human VEGF3 and DNA (RNA) encoding such VEGF3 polypeptides are disclosed. A method for producing such polypeptides is also provided, by recombinant techniques and antibodies and antagonists against such a polypeptide. A method for using these polypeptides to stimulate wound healing and repair of vascular tissue is also disclosed. Methods that use antagonists to inhibit tumor growth, inflammation, and treat diabetic retinopathy, rheumatoid arthritis, and psoriasis are also provided. Diagnostic methods to detect mutations in the sequence encoding VEGF3 and alterations in the concentration of VEGF3 protein in a sample derived from a host, are also known

Description

GROWTH FACTOR 3 ENPOTEL1AL. VASCULAR. HUMAN This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of these polynucleotides and polypeptides. The polypeptide of the present invention has been identified as a member of the vascular endothelial growth factor family. More particularly, the polypeptide of the present invention is vascular endothelial growth factor 3, sometimes referred to hereinafter as "VEGF3". The invention also relates to the inhibition of the action of these polypeptides. The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth and tissue repair. However, angiogenesis is an essential part of certain pathological conditions, such as neoplasia, for example, tumors and gliomas, and 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)). Fibroblast growth factor molecules, both acidic and basic, are mitogenic for endothelial cells and other cell types. Angiotropin and angiogenin can induce angiogenesis, although their functions are unclear. (Folman, J., 1993, Cancer Medicine pp. 153-170, Lea and Fabiger Press). A highly selective mitogen for vascular endothelial cells is the vascular endothelial growth factor or VEGF (Ferrara, H., et al., Endocr. Rev. 13: 19-32 (1992)), also known as the 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 gene of murine VEGF has been characterized by its pattern of expression in embryogenesis and 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 the glomerulation of the kidney. The data are consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation (Breier, G et al., Development, 114: 521-532 (1992)). VEGF is structurally related to the a and ß chains of platelet-derived growth factor (PDGF), a mitogen for mesenchymal cells and placental growth factor (PLGF), a mitogen of endothelial cells. These three proteins belong to the same family and share a conserved motif. Eight cysteine residues, which contribute to the formation of the disulfide bond, are strictly conserved in these proteins. Alternatively, spliced mRNAs have been identified for both VEGF, PLGF and PDGF and these different spliced products differ in biological activity and receptor binding specificity. The function of VEGF and PDGF as homo-dimers or hetero-dimers and the binding to receptors that produce the activity of the intrinsic tyrosine kinase following the dimerization of the receptor. VEGF has four different forms of 121, 165, 189 and 206 amino acids, due to the alternative splicing. VEGF 121 and VEGF 165 are soluble and capable of promoting angiogenesis, while VEGF 189 and VEGF 306 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. Muthukrishnan, L., Wardner, E., D'Amore, P.A., J. Cell. Biol. , 109: 811-822, (1989)). Their high-affinity binding sites are located only in endothelial cells in sections of tissue (Jakeman, L.B., et al., Clin. Invest. 98: 244-253, (1989)). The factor can be isolated from pituitary cells and several lines of tumor cells, and has been implicated in some human gliomas (Piet, K. H. Nature 359: 845-848, (1992)). Interestingly, the expression of VEGF 121 or VEGF 165 gives Chinese hamster ovary cells the ability to form tumors in mice without compensation (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 mice deficient in immunization (Kim, K. J., Na ture 362: 841-844, (1993)). In addition, a dominant negative mutant of the VEGF receptor has been shown to inhibit the growth of glioblastomas in mice. The vascular permeability factor has also been found to be responsible for persistent microvascular hyperpermeability to plasma proteins, even after cessation of injury, 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., 176: 1375-9 (1992). The expression of VEGF is high in vascularized tissues, (for example lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VEGF has also been shown to induce angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissues, especially vascular tissues, VEGF has been proposed for use in promoting repair of vascular tissues (for example in atherosclerosis). U.S. Patent No. 5,073,492, issued December 17, 1991 to Chen et al., Discloses a method for synergistically increasing endothelial cell growth in an appropriate medium, which comprises adding to the medium, VEGF, effectors and the like. factor derived from serum. Likewise, the DNA of the C-cell subunit of vascular endothelial cell growth has been prepared by polymerase chain reaction techniques. DNA encodes a protein that can exist as a hetero-dimer or homo-dimer. The protein is a mammalian vascular endothelial cell mitogen and, as such, is useful for the promotion of vascular development and repair, as described in European Patent Application No. 92302750.2, published on September 30, 1992. The polypeptides of the present invention have been purportedly identified as a novel vascular endothelial growth factor, based on the homology of the amino acid sequence to human VEGF. In accordance with one aspect of the present invention, novel mature polypeptides are provided as well as biologically active and diagnostically and therapeutically useful fragments, their analogs and derivatives. The polypeptides of the present invention are of human origin. According to another aspect of the present invention, isolated nucleic acid molecules encoding the polypeptides of the present invention, including the raRNAs, DNAs, cDNAs, genomic DNA, as well as their biologically active and diagnostically or therapeutically useful fragments, are provided. , its analogues and its derivatives. According to yet another aspect of the present invention, processes for producing such polypeptides are provided, by recombinant techniques, which comprises culturing recombinant prokaryotic and / or eukaryotic host cells, comprising a nucleic acid sequence encoding a polypeptide of the present invention , under conditions that promote the expression of proteins and the subsequent recovery of proteins. In accordance with a further aspect of the present invention, a process for the use of such polypeptides or polynucleotides encoding these polypeptides is provided, for therapeutic purposes, by example, to stimulate angiogenesis, wound healing, and to promote vascular tissue repair.

According to yet another aspect of the present invention, antibodies against such polypeptides are supplied. According to yet another aspect of the present invention, antagonists are provided against such polypeptides and processes for their use in inhibiting the action of these polypeptides, for example to inhibit the growth of tumors, to treat diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis. 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. In accordance with another aspect of the present invention, methods of diagnosing diseases or susceptibility to diseases related to mutations in the nucleic acid sequences of the present invention and the proteins encoding such nucleic acid sequences are provided. According to another aspect of the present invention, a process for the use of such polypeptides, or polynucleotides encoding these polypeptides, is provided for purposes related to scientific research, DNA synthesis and manufacture of DNA vectors. These and other aspects of the present invention will be apparent to those skilled in the art from the teachings of the present invention. The following drawings are only illustrations of specific embodiments of the present invention and do not limit the scope of the invention, which is defined by the claims. Figure 1 illustrates the cDNA sequence and deduced amino acid sequence of the polypeptide of the present invention. Standard one-letter abbreviations for amino acids are used. The sequence is performed using the Automatic DNA Sequencer 373 (Applied Biosystems, Inc.). The accuracy of the sequence is predicted to be greater than 97%. Figure 2 is an illustration of the amino acid sequence homology between the polypeptide of the present invention and human VEGF. According to one aspect of the present invention, isolated nucleic acid molecules (polynucleotides) encoding the mature polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No., May 26, 1995. A polypeptide encoding a polypeptide of the present invention can be obtained from osteoclastomas of the early stage human embryo (week 8 to 9), cell lines of the adult heart or various breast cancers. The polynucleotide of this invention was discovered in a human colon tissue cDNA library. It is structurally related to the VEGF / PDGF family. VEGF3 contains an open reading frame that encodes a protein of 221 amino acid residues. The protein exhibits the highest homology of the amino acid sequence to a human vascular endothelial growth factor with -36.199% identity at 66.063% similarity. It is particularly important that all eight cysteines are conserved within all polypeptides of the present invention and that the identity of the PDGF / VEGF family, PXCVXXXRCXGCCN, is conserved in VEGF3 (see Figure 2). The VEGF3 polypeptide of the present invention includes the polypeptide and the full length polynucleotide sequence, which codes for any leader sequence and for the active fragments of the full length polypeptide. The active fragments include any portion of the full length amino acid sequence, which have less than the full 221 amino acids of the full-length amino acid sequence, as shown in SEQ ID No. 2 and in Figure 1, but still they contain the eight cysteine residues shown conserved in Figure 1 and such fragments will still contain the activity of VEGF3. The polynucleotide of the present invention may be in the form of the RNA or in the DNA form, this DNA includes the cDNA, genomic DNA, and synthetic DNA. The DNA can be double-stranded or single-stranded, and if it is single-stranded, it can be a coding cord or a non-coding (anti-sensitive) cord. The coding sequence encoding the mature polypeptide may be identical to the coding sequence shown in Figure 1 (SEA ID NO: 1) or that of the deposited clone or may be a different coding sequence, this coding sequence, as a result of the redundancy or degeneracy of the genetic key, which encodes the same mature polypeptide as the DNA of Figure 1 (SEQ ID NO: 1) or the deposited cDNA. The polynucleotides encoding the mature polypeptide of Figure 1 (SEQ ID NO: 2) or for the mature polypeptides encoded by the deposited cDNAs may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally the additional coding sequence) and the non-coding sequence, such as the introns or the 5 'and / or 3' non-coding sequence of the coding sequence for the mature polypeptide. Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only the coding sequence for the polypeptide as well as a polynucleotide which includes the additional coding sequence and / or non-coding sequence. The present invention furthermore relates to variants of the polynucleotides described above, which encode the fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or the polypeptides they encode. the cDNA of the deposited clone (s). The variants of the polynucleotides may be naturally occurring allelic variants of the polynucleotide or a non-naturally occurring variant of the polynucleotide. Thus, the present invention includes polynucleotides that encode the same mature polypeptide as shown in Figure 1 (SEQ ID NO: 2) or the same mature polypeptides encoded by the cDNA (s) of the deposited clones, as well as variants of such polynucleotides, these variants encode a fragment, derivative or analogue of the polypeptide of Figure 1 (SEQ ID NO: 2) or the polypeptides encoded by the cDNA (s) of the deposited clones. These nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As indicated hereinabove, the polynucleotide may have a coding sequence, which is an allelic variant that occurs naturally from the coding sequence shown in Figure 1 (SEQ ID NO: 1) or from the coding sequence of the (s) clone (s) deposited (s). As is known in the art, an allelic variant is an alternative form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which do not substantially alter the function of the encoded polypeptides. The polynucleotides of the present invention may also have the coding sequence fused to a marker sequence, which allows the purification of the polypeptide of the present invention. The marker sequence may be a hexahistidine tag supplied by a pQE-9 vector, to provide for the purification of the mature fused polypeptide to the tag in the case of a bacterial host or, for example, the tag sequence may be a hemagglutinin tag (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, I., et al., Cell, 37: 767 (1984)). The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes the regions that precede and follow the coding region (guide and drag) as well as the intervening sequences (introns) between the individual coding segments (exons). Fragments of the full-length gene can be used as a hybridization probe for a cDNA library to isolate the full-length cDNA and to isolate other cDNAs that have a high sequence similarity to the gene or a 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 that corresponds to a full-length transcript and a genomic clone or clones, which contain the entire gene, which includes regulatory and promoter regions, exons and introns. An example of a selection comprises isolating the coding region of the gene using the known DNA sequence to synthesize an oligonucleotide probe. The labeled oligonucleotides, which have a sequence complementary to that of the gene of the present invention, are used to select a collection of the human cDNA, the genomic DNA or mRNA, to determine which members of the collection hybridize the probe. The present invention further relates to polynucleotides that hybridize to the sequences described above, if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides that hybridize under severe conditions to the polynucleotides described herein above. As used herein, the term "severe conditions" means that hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides that hybridize to the polynucleotides, hereinbefore described herein, in a preferred embodiment, encode polypeptides that retain substantially the same function or biological activity as the mature polypeptide encoded by the cDNAs of Figure 1 (SEQ ID N0: 1) or the cDNAs deposited. Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases and more preferably at least 50 bases, which hybridize to a polynucleotide of the present invention and which have an identity there, as described above, and which may or may not retain the activity. For example, each polynucleotide can be used as a probe for the polynucleotide of SEQ ID NO: 1, for example, to recover the polynucleotide or as a diagnostic probe or as a polymerase chain reaction (PCR) primer. A) Yes, the present invention is directed to polynucleotides having at least 70% identity, preferably at least 90% and more preferably at least 95% identity, to a polynucleotide encoding the polypeptide of SEQ ID NO: 2, like their fragments, these fragments have at least 30 bases and preferably at least 50 bases and the polypeptides encoded by these polynucleotides. The deposits mentioned here will be kept under the Budapest Treaty in the International Recognition of the Deposit of Microorganisms for the purposes of the Patent Procedure. These deposits are provided merely as a convenience and not to admit that a deposit is required, according to Code 35 U.S.C. § 112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded herein, are incorporated by reference and are the control in the case of any conflict with the description of the sequences herein. . A license may be required to obtain, use or sell the deposited materials and no license is granted hereby. The present invention furthermore relates to a polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2) or having the amino acid sequence encoded by the deposited cDNA (s), as well as the fragments, analogs and derivatives of these polypeptides.

The terms "fragments", "derivatives" and "analogs", when referring to the polypeptide of Figure 1 (SEQ ID NO: 2) or those encoded by the deposited cDNA (s), mean polypeptides that retain the conserved motif of the VEGF proteins, as shown in Figure 1 (SEQ ID NO: 2) and essentially the same function or biological activity. The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides. The fragments, derivatives or analogs of the polypeptide of Figure 1 (SEQ ID NO: 2) or those encoded by the deposited cDNA (s), can be (i) that in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such a substituted amino acid residue may or may not be encoded by the genetic key or (ii) that one or more of the amino acid residues include a substituent group or (iii) that in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (e.g., polyethylene glycol) or (iv) that in which additional amino acids are fused to the mature polypeptide or (v) one in which it comprises fewer amino acid residues than those shown in SEQ ID NO: 2 and retains the conserved motif and still retains the characteristic activity of the VEGF family of s polypeptides. Such fragments, derivatives and the like are considered to be within the scope of those skilled in the art taking into account the present teachings. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form and preferably are purified to homogeneity. The term "isolated" means that the material is removed from its original environment (for example the natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but this same polynucleotide or DNA or polypeptide, separated from it or from all materials coexisting in the natural system, is isolated. These polynucleotides can be part of a vector and / or such polynucleotides or polypeptides can be part of a composition and still be isolated in that that vector or composition is not part of their natural environment. The polypeptides of the present invention include the polypeptide of SEQ ID NO: 2 (in particular the mature polypeptide) as well as polypeptides having at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO: 2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO: 2 and even more preferably at least 95% similarity (further preferably, at least 95% identity) to the polypeptide of SEQ ID NO: 2 and also include portions of such polypeptides with such a portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. As known in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of a polypeptide to the sequence of a second polypeptide. Fragments or portions of the polypeptides of the present invention can be used to produce the corresponding full-length polypeptide by the synthesis of peptides; 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 full-length polynucleotides of the present invention. The present invention also relates to vectors that include the polynucleotides of the present invention, the host cells that are genetically treated with the vectors of the invon and the production of the polypeptides of the invon by recombinant techniques. The host cells can be genetically treated (transduced or transformed or transfected) with the vectors of this invon, which can 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. The treated host cells can be cultured in convonal nutrimedia, modified as appropriate, to activate the promoters, select the transformants or amplify the VEGF genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and this will be apparto one of ordinary skill in the art. The polynucleotides of the presinvon can be used to produce polypeptides by recombinant techniques. Thus, for example, the polynucleotide can be included in any of a variety of expression vectors, to express a polypeptide. Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, eg, SV40 derivatives; bacterial plasmids; Phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccines, adenoviruses, virus of postulose eruptions of birds and pseudo-rabies. However, any other vector can be used as long as it is duplicable and viable in the host. The appropriate sequence of the DNA can be inserted into the vector by a variety of procedures. 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. The DNA sequence in the expression vector is operably linked to one or more appropriate expression control sequences (promoters) to direct the synthesis of the mRNA. As representative examples of such promoters, there may be mentioned, the LTR or SV40 promoter, E. coli, lac or trp, the PL promoter from phage lambda and other known promoters in the control of gene expression in prokaryotic or eukaryotic cells or their virus. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for the expression of amplification. In addition, expression vectors preferably contain a gene for providing a phenotypic treatment for the selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or resistance to tetracycline or ampicillin. in E. coli. The vector containing the appropriate DNA sequence, as described above, as well as an appropriate promoter or control sequence, can be employed to transform an appropriate host to allow the host to express the protein. As representative examples of appropriate hosts, there may be mentioned bacterial cells, such as E. coli, Salmonella typhimurium,; fungal cells, such as yeasts; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or Bowes melanoma; adenovirus; plant cells, etc. The selection of an appropriate host is considered to be within the scope of those skilled in the art, of the present teachings. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as described broadly above. The builders comprise a vector, such as a plasmid or a viral vector, in which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, which include, for example, a promoter, operably linked to the sequence.

Large numbers of suitable vectors and promoters are known to those skilled in the art, and are commercially available. The following vectors are provided in the form of an example. Bacteria: pQR70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNHSa, pNH16a, pNH18a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: p Lneo, pSV2cat, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector can be used as long as it is duplicable and viable in the host. 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 immediate primary DMV, HSV thymidine kinase, primary and late SV40, retrovirus LTRs and mouse metallothionein-I. The selection of the appropriate vector and promoter is well within the level of ordinarily skilled in the art. In a further embodiment, the present invention relates to host cells containing the above-described manufacturer. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a minor eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The introduction of the builder into the host cell can be effected by transfection of calcium phosphate, transfection mediated by DEAE-Dextran, or electroporation (Davis, L., Dibner, M. Battey, I., Basic Methods in Molecular Biology, (1986)). The builders in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be produced synthetically by conventional peptide synthesizers. 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 employed to produce these proteins, using the RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Labora tory Manual, Second Edition (Cold Spring Harbor, NY, 1989), the disclosure of which is incorporated herein. as reference.

The transcription of a DNA encoding the polypeptides of the present invention by higher eukaryotes is enhanced by inserting an enhancer sequence into the vector. Enhancers are the cis-active elements of DNA, usually around 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the latter side of the replication origin (bp 100 to 270), an early cytomegalovirus promoter enhancer, a polyoma enhancer on the latter side of the replication origin and the adenovirus enhancers. Generally, recombinant expression vectors will include replication origins and selectable markers that allow transformation of the host cell, for example, the E. coli ampicillin resistance gene and the S TRPI gene. cerevisiae and a promoter derived from a gene highly expressed to direct the transcription of a downstream structural sequence. Such promoters can be derivatives of glycolytic enzymes that code operons, such as 3-phosphoglycerate kinase (PGK), factor a, acid phosphates or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with the translation sequences, initiation and termination, and preferably, a guide sequence, capable of directing the secretion of the translated protein in the periplasmic space or the extracellular environment. Optionally, the heterologous sequence can encode a fusion protein, which includes an N-terminal identification peptide, which imparts the desired characteristics, for example stabilization or simplified purification of the expressed recombinant product. Useful vectors of expression for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein, together with suitable translational, initiation and termination signals in the operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and a replication origin to ensure maintenance of the vector and, if desired, to provide amplification within the host. Prokaryotic hosts suitable for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and several species within the genera of Pseudomonas, Streptomyces and Staphilococcus, although others may also be used as selection material. As a representative, but non-limiting example, expression vectors useful for bacterial use may comprise a selectable marker and the bacterial origin of replication derived from commercially available plasmids, comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017), Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMI (promete Biotec, Madison, Wl, USA). These "skeleton" sections pBR322 are combined with an appropriate promoter and the structure sequence will be expressed. Following transformation of the appropriate host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for a period of time. additional. The cells are typically collected by centrifugation, disruption 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 broken by any convenient method, which includes the freeze-melt cycle, sonication, mechanical disruption, or use of cell lysis agents, such methods are well known to those skilled in the art. . Several mammalian cell culture systems can also be employed to express the recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127 cell lines. , 3T3, CHO, HeLA and BHK. Mammalian expression vectors will comprise a replication origin, a suitable promoter and enhancer, and also any necessary binding site of the ribosome, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences and non-transcribed flanking sequences 5'. DNA sequences derived from the SV40 viral splice and polyadenylation sites can be used to deliver the required non-transcribed genetic elements. The polypeptides of the present invention can be recovered and purified from recombinant cell cultures by methods that include ammonium sulfate or ethanol precipitation, acid extraction, anion exchange or cation chromatography, phosphocellulose chromatography, hydrophobic interaction, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Protein redoubling steps can be used, as necessary, to complete the mature protein configuration. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step. The polypeptides of the present invention can be a naturally purified product or a product of synthetic chemical processes or produced by the recombinant techniques of a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammals, in the crop). Depending on the host employed in the recombinant production method, the polypeptides of the present invention may be glycosylated or may not be glycosylated. The polypeptides of the invention may also include an initial methionine amino acid residue. The VEGF3 polypeptide can be employed in promoting angiogenesis, for example, to stimulate the growth of transplanted tissue, when performing coronary bypass surgery. VEGF3 can also be employed in stimulating wound healing, particularly for revascularizing damaged tissues or where new capillary angiogenesis is desired. VEGF3 can be used to treat full thickness wounds, such as dermal ulcers, which include venous ulcers, irritable pressure, and diabetic ulcers. In addition, VEGF3 can be used to treat burns and full thickness injuries, where a skin graft or fin is used to repair these burns and injuries. VEGF3 can also be employed for use in plastic surgery, for example for the repair of lacerations of traumas and cuts associated with surgery. Along with these same lines, VEGF3 can be used to induce the growth of damaged bones, tissue of the periodontium or ligament. VEGF3 can also be used to regenerate supporting tissues of the teeth, which include the demention and periodontal ligaments, which have been damaged by disease and trauma. Since angiogenesis is important in keeping wounds clean and non-infectious, VEGF3 can be used in association with surgery and after repair of cuts. It can also be used in the treatment of abdominal wounds, where there is a high risk of infection. VEGF3 can be used for the promotion of endothelialization in vascular graft surgery. In the case of vascular grafts using the transplanted or synthetic material, VEGF3 can be applied to the surface of the graft or to the joints to promote the growth of vascular endothelial cells. VEGF3 can also be used in the repair of myocardial tissue damage, as a result of myocardial infarction. VEGF3 can also be used to repair the cardiac vascular system after ischemia. VEGF3 can also be used to treat damaged vascular tissues, as a result of coronary artery disease and peripheral and vascular CNS disease. VEGF3 can also be used to coat artificial prostheses or natural organs, which are going to be transplanted in the body, to minimize the rejection of the transplanted material and stimulate the vascularization of the transplanted materials. VEGF3 can also be used for the repair of vascular tissue, for example, which requires during arteriosclerosis and after angioplasty of distension, where the vascular tissues are damaged. The nucleic acid sequences of VEGF3 and the VEGF3 polypeptides can also be used for in vi tro purposes, related to scientific research, DNA synthesis and manufacture of DNA vectors and for the production of diagnostics and therapies to treat human diseases. For example, VEGF3 can be used for the in vitro culture of vascular endothelial cells, where it is added to the conditional medium in a concentration of 10 pg / ml up to 10 ng / ml. This invention provides methods for the identification of VEGF3 receptors. The gene that encodes the receptor can be identified by numerous methods, known to those skilled in the art, for example, ligand and FACS classification (Coligan et al., Current Protocols in Immun., 1 (2), Chapter 5, (1991)). Preferably, expression cloning is employed where polyadenylated RNA is prepared from a VEGF3 responsive cell and a cDNA library created from this RNA is divided into groups and used to transfect COS cells or other cells that are not sensitive to VEGF3 . Transfected cells growing on glass slides are exposed to labeled VEGF3. This VEGF3 is labeled by a variety of resources, including the iodization or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. The positive groups are identified and the sub-groups are prepared and retransfected using an iterative subgroup and a reclassification process, finally providing a simple clone that encodes the supposed recipient. As an alternative approach for receptor identification, tagged VEGF3 can be linked by photoaffinity with cell membrane preparations or extract expressing the receptor molecule. The interlaced material is resolved by PAGE electrophoresis and exposed to an X-ray film. The labeled complex containing VEGF3 is then excised, resolved into peptide fragments and subjected to protein microequence. The amino acid sequence obtained from the microsequence will be used to design a set of degeneration oligonucleotide probes, to classify a collection of lac DNA to identify the gene encoding the putative receptor. This invention also relates to a method for classifying compounds to identify those that are agonists or antagonists of VEGF3. An example of such a method takes advantage of the ability of VEGF3 to significantly stimulate the proliferation of human endothelial cells, in the presence of the A-comitogen. The endothelial cells are obtained and cultured in 96-well flat bottom culture dishes ( Costar Cambridge, MA), in a reaction mixture supplemented with Con-A (Calbiochem, La Jolla, CA). The Con-A polypeptides of the present invention and the compound to be classified are added. After incubation at 37 ° C, cultures are pulsed with 1 μCi of 3 [H] thymidine (5 Ci / mmol; 1 Ci = 37 BGq; NEN) for a sufficient time to incorporate the 3 [H] and collect it in glass fiber filters (Cambridge Technology, Watertown, MA). The average incorporation of ^ [H] thymidine (cpm) from triplicate cultures was determined using a liquid scintillation counter (Beckman Instrumnents, Irvine, CA). The significant incorporation of 3 [H] -thymidine, compared to a control assay, where the compound is excluded, indicates the stimulation of endothelial cell proliferation.

For testing the antagonists, the assay described above is performed and the ability of the compound to inhibit the incorporation of 3 [H [-thymidine, in the presence of VEGF3 indicates that the compound is an antagonist to VEGF3. Alternatively, VEGF3 antagonists can be detected by combining VEGF3 and a potential antagonist with membrane-bound VEGF3 receptors or combining receptors under conditions appropriate for a competitive inhibition assay. VEGF3 can be labeled, such as by radioactivity, so that the number of VEGF3 molecules bound to the receptor can determine the effectiveness of the potential antagonist. Alternatively, the response of a second messenger system known at once to the interaction of VEGF3 and the receptor is measured and compared in the presence or absence of the compound. These second messenger systems include, but are not limited to, the cAMP guanylate cyclase, the ion channels or the hydrolysis of the phosphoinositidine. In another method, a mammalian cell or a membrane preparation expressing the VEGF3 receptor is incubated with the labeled VEGF3, in the presence of the compound. The ability of the compound to intensify or block this interaction can then be measured. Potential VEGF3 antagonists include an antibody or, in some cases, an oligonucleotide, which. they bind to the polypeptide and effectively eliminate the function of VEGF3. Alternatively, a potential antagonist may be a closely related protein, which binds to the VEGF3 receptors, however, they are inactive forms of the polypeptide and thus prevent the action of VEGF3. Examples of these antagonists include a dominant negative mutant of the VEGF3 polypeptide, for example, a strand of the hetero-dimeric form of VEGF3 may be dominant and may be subject to mutation so that biological activity is not retained. An example of a negative dominant mutant includes truncated versions of the dimeric VEGF3, which is capable of interacting with another dimer to form a wild-type VEGF3, however, the resulting homo-dimer is not active and fails to exhibit the characteristic activity of VEGF. . Another potential antagonist of VEGF3 is an antisensitive construct, prepared using the antisensitive type technology. This technology can be used to control the expression of the gene through triple helix formation or antisensitive DNA or RNA, both methods are based on the binding of a polynucleotide to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence, which codes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. An oligonucleotide of DNA is designed to be complementary to the region of the gene involved in transcription (triple helix, see Lee et al., Nucí Acids Res., 6: 3073 (1979); Cooney et al., Science, 241: 456 (1938) and Dervan et al., Science 251: 1360 (1991)). thus preventing the transcription and production of VEGF3. The antisense RNA oligonucleotide hybridizes to mRNA in vivo and blocks the translation of the mRNA molecule into the VEGF3 polypeptide (Antisense-Okano, J. Neurochem, 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press , Boca Ratón FL (1988)). The oligonucleotides, described above, can also be delivered to the cells, so that the antisensible RNA or DNA can be expressed in vivo to inhibit the production of the polypeptide. Potential VEGF3 antagonist compounds also include small molecules that bind to and occupy the ligand site of the receptors, thereby rendering the receptor inaccessible to the substrate, so that normal biological activity is impeded. Examples of small molecules include, but are limited to, small peptides or peptide-like molecules. Antagonist compounds can be used to treat tumors, since angiogenesis and neovascularization are essential stages in tumor growth. The mRNA encoding VEGF3 was found to be expressed at moderate levels in at least two breast tumor cell lines, which is indicative of the role of VEGF3 polypeptides in the malignant phenotype. Gliomas are also a type of neoplasm that can be treated with the antagonists of the present invention. These antagonists can also be used to treat inflammations caused by increased vascular permeability. In addition to these disorders, antagonists can also be used to treat diabetic retinopathy, rheumatoid arthritis and psoriasis. Antagonists can be employed in a composition with a pharmaceutically acceptable carrier, for example as described below. The VEGF3 polypeptides and agonists and antagonists can be used in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to, a saline solution, regulated saline solution, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation must be suitable for the mode of administration.

The invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with these containers may be a notification in the form prescribed by the government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, this notification reflects the approval by the manufacturing, use or sale agency for human administration. In addition, the polypeptides, agonists and antagonists of the present invention can be used in conjunction with other therapeutic compounds. The pharmaceutical compositions can be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositions are administered in an amount that is effective for the treatment and / or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg / kg of body weight and, in many cases, they are administered in an amount of no more than about 8 mg / kg of body weight per day. In many cases, the dose is approximately 10 μg / kg to 1 mg / kg of body weight per day, taking into account the route of administration, symptoms, etc.

The polypeptide of the invention and the agonist and antagonist compounds, which are polypeptides, can also be used according to the present invention for the expression of such polypeptides in vivo, which is often referred to as "gene therapy". Thus, for example, cells, such as bone marrow cells, can be treated with a polynucleotide (DNA or RNA) encoding the polypeptide ex vivo, the treated cells are then delivered to a patient to be treated with the polypeptide. These methods are well known in the art. For example, cells can be treated by methods known in the art, by the use of RNA containing a retroviral particle encoding the polypeptide of the present invention. Similarly, cells can be treated in vivo for expression of the polypeptide in vivo, for example by methods known in the art. As is known, a producer cell, to produce the RNA containing - a retroviral particle, which encodes the polypeptide of the present invention, can be administered to a patient to treat the cells in vivo and the expression of the polypeptide in vivo These and other Methods of administering the polypeptide of the present invention by such methods will be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for the treated cells may be other than a retroviral particle, for example, an adenovirus, which can be used to treat the cells in vivo prior to combination with a suitable delivery vehicle. Retroviruses from which the aforementioned retroviral plasmid vectors can be derived, include, but are not limited to, Moloney Virus from Murine Leukemia, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus. , Harvey's sarcoma virus, bird leukosis virus, gibbon simian leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus and virus, mammary tumor. In one embodiment, the retroviral plasmid vector is derived from the Moloney Virus of Murine Leukemia. The vector includes one or more promoters. Suitable promoters that may be employed include, but are not limited to, retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter, described by Miller et al., Biotechniques Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters, such as eukaryotic cell promoters). , including, but not limited to, histone, pol III, and β-actin promoters). Other viral promoters that may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and parvovirus B19 promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the tengs contained herein. The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter.; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the promoter of albumin; the ApoAI promoter; the promoters of human globin; promoters of the viral thymidine kinase, such as the Herpes Simplex thymidine kinase promoter; Retroviral LTRs (including modified retroviral LTRs, described above); the β-actin promoter; and the promoters of human growth hormone. The promoter may also be the native promoter that controls the gene encoding the polypeptide. The retroviral plasmid vector is used to transduce the packaging cell lines to form the cell lines of the producer. Examples of packaging cells that can be transfected include, but are not limited to, the cell lines PE501, PA317,? -2,? -AM, PA12, T19-14X, VT-19-17-H2,? CRE, ? -CRIP, GP + E + 86, GP + envAml2 and DAN, as described in Miller Human Gene Theory, Vol. 1, pages 5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. These means include, but are not limited to, electroporation, the use of liposomes, and the precipitation of CaP04. In an alternative, the retroviral plasmid vector may 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 one or more nucleic acid sequences, which encode the polypeptides. Such retroviral vector particles can then be employed, to transduce the eukaryotic cells, or in vi tro or in vivo. The transduced eukaryotic cells will express one or more nucleic acid sequences encoding the polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells and bronchial epithelial cells.

This invention also relates to the use of VEGF3 genes as part of a diagnostic assay for detecting diseases or susceptibility to diseases related to the presence of mutations in the VEGF3 nucleic acid sequences encoding the polypeptide of the present invention. . Individuals that carry mutations in a VEGF3 gene can be detected at the DNA level by a variety of techniques. The nucleic acids for diagnosis can be obtained from the patient's cells, such as blood, urine, saliva, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by the use of PCR (Saiki et al., Na ture, 324: 163-166 (1986)) before analysis. The RNA or the cDNA can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding a polypeptide of the present invention can be used to identify and analyze the mutations. For example, deletion and insertions can be detected by a change in the size of the amplified product, compared to the normal genotype. Peak mutations can be identified by hybridization of amplified DNA to radiolabeled RNA or alternatively, radiolabelled anti-sensitive DNA sequences. The sequences that correspond perfectly can be distinguished from duplexes not in correspondence by the digestion of the RNase A or by differences in the fusion temperatures. Genetic testing based on DNA sequence differences can be achieved by the detection of * alteration in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Suppressions and insertions of small sequences can be visualized by high resolution gel electrophoresis. DNA fragments from different sequences can be distinguished from the gradient gels of denatured formamide in which the mobilities of the different DNA fragments are delayed in the gel at different positions, according to their specific melting temperatures or partial melting. (see, for example, Myers et al., Science, 230: 1242 (1985)). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as the protection of RNase and SI or the chemical cleavage method (eg, Cotton et al., PNAS, USA, 85: 4397-4401 (1985)). Thus, the detection of a specific DNA sequence can be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequence or the use of restriction enzymes (for example, Fragment Length Polymorphisms). of Restriction (RFLP)) and Southern blotting of genomic DNA. In addition to the more conventional gel electrophoresis and the DNA sequence, mutations can also be detected by in situ analysis. The present invention also relates to a diagnostic assay for detecting the altered levels of VEGF3 proteins in various tissues, since an over-expression of the proteins, compared to normal control tissue samples, can detect the presence of the Abnormal cell proliferation, for example, a tumor. The assays used to detect the levels of the protein in a sample derived from a host are well known to those skilled in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, ELISA assays and "sandwich assay" An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1 (2), Chapter 6, (1991)), comprises initially preparing an antigen-specific antibody to the polypeptides of the present invention, preferably a monoclonal antibody.In addition to a reporter antibody is prepared against the monoclonal antibody.To the reporter antibody is attached a detectable reagent, such as radioactivity, fluorescence or, in this example, a strong horseradish peroxidase enzyme. A sample is removed from a host and incubated on a solid support, for example a polystyrene disk, which binds the proteins in the sample. Any free protein binding site on the disk is then covered by incubation with a non-specific protein, such as bovine serum albumin. Next, the monoclonal antibody is incubated on the disc during this time the monoclonal antibodies bind to any VEGF3 protein attached to the polystyrene disk. All unbound monoclonal antibody is washed and separated with a buffer solution. The reporter antibody bound to horseradish peroxidase is now placed on the disk, which results in the binding of the reporter antibody to any monoclonal antibody bound to VEGF3. The reporter antibody not attached is then washed and separated. The peroxidase substrates are then added to the disk and the amount of color developed in a given period of time is a measurement of the amount of the VEGF3 protein present in a given volume of the patient sample, when compared to a standard curve. . A competition assay can be employed, in which the VEGF3 specific antibody binds to a solid support. The polypeptides of the present invention are then labeled, for example by radioactivity, and a sample derived from the host is passed over the solid support and the amount of label detected, for example, by liquid scintillation chromatography, can be correlated to a amount of VEGF3 in the sample. A "sandwich" trial is similar to a trial ELISA In a "sandwich" assay VEGF3 is passed over a solid support and binds to the attached antibody to a solid support. A second antibody then binds to VEGF3. A third antibody, which is labeled and specific to the second antibody, is then passed over the solid support and bound to the second antibody and an amount can then be quantified. The sequences of the present invention are also valuable for the identification of chromosomes. The sequence is specifically the target and can be hybridized to a particular location on an individual human chromosome. Also, there is a current need to identify particular sites on the chromosome. Few chromosome labeling reagents, based on the actual sequence data (polymorphism repeat) are currently available to mark the location of chromosomes. The mapping of the DNA to the chromosomes, according to the present invention, is a first important step in correlating these sequences with the genes associated with diseases.

In brief, the sequences can be mapped to chromosomes by preparing the PCR primers (preferably 15 to 25 bp) of the cDNA. Computer analysis of the 3 'untranslated region is used to quickly select the primers, so that they do not extend for more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for the classification of hybrid somatic cell PCRs containing individual human chromosomes. Only those hybrids that contain the human gene that correspond to the initiator will supply an amplified fragment. The mapping of the somatic cell hybrid PCR is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, the sub-location can be accomplished with panels of specific chromosome fragments or groups of large genomic clones in an analogous manner. Other mapping strategies that can be used similarly for mapping your chromosome include hybridization in itself, previously classified with labeled labeled fluxes and pre-selection by hybridization to build collections of the chromosome-specific cDNA. Fluorescence in-situ hybridization (FISH) from a cDNA clone to a metaphase chromosomal extension can be used to deliver a precise chromosomal location in one step. This technique can be used with a cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes, a Manual of Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available online through Johns Hopkins University Welch Medical Library.) The relationship between genes and diseases that have been mapped to some chromosomal region are Then, it is necessary to determine the differences in the cDNA or the genomic sequence between affected and unaffected individuals, if a mutation is observed in some or all of the individuals. affected, but not in a normal individual, then the mutation is probably the causative agent of the disease.With the current resolution of the techniques of physical mapping and genetic mapping, a cDNA located precisely in a chromosomal region associated with the disease, can be one of between 50 and 500 potential cause genes. (This supposes a mapping resolution of 1 megabase and one gene per 20 kb). olipeptides, their fragments or other derivatives, or their analogues, or cells expressing them, they can be used as an immunogen to produce antibodies. These antibodies can be, for example, polyclonal or monoclonal. The present invention also includes chimeric single chains and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various methods known in the art can be used for the production of such antibodies and fragments. Antibodies raised against polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administration of the polypeptides to an animal, preferably non-human. The antibody, thus obtained, will then bind to the polypeptides themselves. In this way, even a sequence encoding only a fragment of the polypeptides can be used to generate antibody that bind to the total native polypeptides. Such antibodies can then be used to isolate the polypeptide from the tissue expressing that polypeptide. For the preparation of monoclonal antibodies, any technique that delivers antibodies produced by continuous cultures of cell lines can be used. Examples include the hybridoma technique (Kohier and Milstein, 1975, Na ture, 256: 495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72). and the EBV hybridoma technique, to produce human monoclonal antibodies (Colé et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pages 77-96). The technique described for the production of single chain antibodies (U.A. Patent No. 4,946,778) can be adapted to produce single chain antibodies to the immunogenic polypeptide products of this invention. Likewise, transgenic mice can be used to express humanized antibodies to the immunogenic polypeptide products of this invention. The present invention will be further described with reference to the following examples; however, it will be understood that the present invention is not limited to such examples. All parts or quantities, unless otherwise specified, are by weight. In order to facilitate understanding of the following examples, certain methods that occur frequently and / or terms will be described.

"Plasmids" are designated by a lowercase letter p preceded and / or followed by capital letters and / or numbers. The starting plasmids here are commercially available, with advertising available on an unrestricted basis, or plasmids can be constructed available, according to published procedures. In addition, plasmids equivalent to those described are known in the art and will be apparent to those of ordinary skill in the art. "Digestion" of DNA refers to the catalytic cleavage of DNA with a restriction enzyme, which acts only in certain sequences in DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements are used as will be known to those of ordinary skill in the art. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of the buffer. For the purpose of isolating DNA fragments for the construction of plasmids, typically 5 to 50 μg of the DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate amounts of regulators and substrates for the particular restriction enzymes are specified by the manufacturer. Incubation times of approximately 1 hour at 37 ° C are those used ordinarily, but may vary according to the instructions of the supplier. After digestion, the reaction is subjected directly to electrophoresis in a polyacrylamide gel to isolate the desired fragment. The size separation of the split fragments is carried out using 8 percent of the polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res. , 8: 4057 (1980). "Oligonucleotides" refer to any single-stranded polydeoxynucleotide or two strands of complementary polydeoxynucleotides, which can be chemically synthesized. Such synthetic oligonucleotides do not have 5'-phosphate and thus do not bind to another oligonucleotide without adding a phosphate with ATP in the presence of a kinase. A synthetic oligonucleotide will be ligated to a fragment that has not been dephosphorylated. "Ligation" refers to the process of forming phosphodiester bonds between two double-stranded nucleic acid fragments (Maniatis, T., et al., Id., P.146). Unless otherwise mentioned, ligation can be accomplished using known regulators and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

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

EXAMPLE 1 Cloning and Expression of VEGF3 with the Use of the Baculovirus Expression System The DNA contained in the deposited clone encoding the VEGF3 protein was amplified using the oligonucleotide primers of the PCR corresponding to the 5 'and 3' sequences of the gene: The 5 'primer has the sequence 5' GCATGGATCCCAGCCTGA TGCCCCTGGCC (SEQ ID NO: 4) and contains, a BamH1 restriction enzyme site and a nucleotide sequence complementary to the 5 'sequence of VEGF3 (nt. 150. 166). The 3 'initiator of the sequence 5' GCATTCTAGACCCTGCTGAG TCTGAAAAGC 3 '(SEQ ID NO: 5) and contains the cleavage site for restriction endonucleases XBal and nucleotides complementary to the 3' sequence of VEGF3. 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 BamHl and Xbal endonucleases and purified again on a 1% agarose gel. This fragment was ligated to the baculovirus transfer vector A2GP with the sequence of guide peptides (PHarmingen) at the BamHl and Xbal sites. Through this link, the VEGF of the cDNA was cloned into the frame with the signal sequence of the gp67 baculovirus gene and placed at the 3 'end of the signal sequence in the vector. This was designated pA2GP-VEGF3. To clone VEGF3 with the gp67 gene signal sequence to the pRG1 vector for expression, VEGF3 with the signal sequence and some upstream sequence were excised from plasmid pA2GP-VEGF3 at the restriction endonuclease site placed upstream - from VEGF3 of the cDNA, and at the restriction endonuclease site of Xbal by the restriction enzyme Xbol and Xbal. This fragment was separated from the rest of the vector in 1% agarose gel and purified with the use of the "Geneclean" equipment. It was designated F2. The A2PG vector (modification of vector pVL941) "was used for the expression of the VEGF3 protein, which uses the baculovirus expression system (for review, see: Summmers, MD and Smith, G E. 1987, A Manual Of Methods For Baculovirus Vectors and Insect Cell Cul ture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555) This expression vector contains the strong polyhedrin promoter of nuclear polyhedrosis virus Autographa californica (AcMNPV), followed by recognition sites for endonucleases BamHI, Smal, Xbal, BglII, and Asp718 restriction sites A site for the Xhol restriction endonuclease is located upstream from the BamHI site The sequence between the XhoI and BamHI sites is the same as that of the pAcGp67A vector. Simian virus (SV) 40 was used for efficient polyadenylation.For an easy relation of the recombinant virus the E. coli beta-galactosidase gene was inserted into the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences were flanked on both sides by viral sequences for the cell-mediated homologous recombination of the co-transfected wild-type viral DNA. Many other baculovirus vectors can be used in place of pA2, such as pRG1, pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170: 31-39). The plasmid was digested with the restriction enzymes and dephosphorylated using the calf intestinal phosphatase by procedures known in the art. The DNA was then isolated from the 1% agarose gel using commercially available equipment ("Geneclean" BIO 101 Inc., La Jolla ¡, Ca.). This vector DNA is designated V2. The F2 fragment and the dephosphorylated V2 plasmid were ligated with the T4 DNA ligase. E. coli HB101 cells are then transformed and the identified bacteria containing the plasmid (pBac gp67-VEGF3) with the VEGF3 gene with the use of the enzymes BamHl and Xbal. The sequence of the cloned fragment was confirmed by the DNA sequence. 5 μg of pBac A2GF-VEGF3 of plasmid was co-transfected with 1.0 μg of a commercially available linearized baculovirus ("BaculoGold® baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Felgner et al. Nati, Acad. Sci. USA, 84: 7413-7417 (1987)). 1 μg of the BaculoGold® virus DNA and 5 μg of the plasmids, in each case, were mixed in well sterilized microtiter plates, containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD). Next, 10 μl of Lipofectin plus 90 μl of Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added in drops to the Sf9 insect cells (ATC CRL 1711) seeded in 35 mm tissue culture plates with 1 ml Grace's medium without serum. The plates were oscillated back and forth to mix the newly added solution. The plates were then incubated for 5 hours at 27 ° C. After 5 hours, the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum were added.

Plates were placed back in an incubator and culture was continued at 27 ° C for four days. After four days, the supernatant was collected and plaque assays performed similarly as described by Sumares and Smith (supra). As a modification, an agar gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was used, which allowed easy isolation of the blue-stained plates. (A detailed description of a "plaque assay" can also be found in the user guide for the cultivation of insect cells and baculovirology distributed by Life Technologies Inc., Gaithersburg, pages 9-10). Four days later, the serial dilution of the virus was added to the cells and the blue-stained plates were collected with the tip of an Eppendorf pipette. The agar containing the recombinant viruses was then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect the Sf9 cells seeded on 35 mm discs. Four days later, the supernatants of these culture discs were collected and then stored at 4 ° C. The Sf9 cells grew in Grace's medium, supplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-A2GP-VEGF3 at a multiplicity of infections (MOI) of 1. Six hours later, the medium was removed and replaced with the SF900 II medium minus methionine and cysteine (Life Technologies Inc. , Gaithersburg). 42 hours later, 5 μCi of the 35s-methionine and 5 μCi of the 35s-cysteine (Amersham) were added. The cells were further incubated for 16 hours before being harvested by centrifugation and the labeled proteins were visualized by SDS-PAGE and autoradiography. The protein of the medium and the cytoplasm of the Sf9 cells were analyzed by SDS-PAGE electrophoresis under reducing and nonreducing conditions. The medium was dialyzed against 50 mM of MES, pH of 5.8. The precites were obtained after dialysis and resuspended in 100 mM Na Citrate, pH 5.0. The resuspended pellet was analyzed again by SDS-PAGE and stained with the Coomassie Brilliant Blue dye. The supernatant of the medium was also diluted 1:10 in 50 mM of MES, pH of 5.8 and applied to a SP-650M column (1.0 x 6.6 cm, Toyopearl) at a flow rate of 1 ml / min. The protein was eluted with gradients of steps at 200, 300 and 500 mM NaCl. VEGF3 was obtained using the elution at 500 mM. The eluate was analyzed by SDS-PAGE electrophoresis in the presence or absence of a reducing agent, β-mercaptoethanol and stained with the Coommassie Brilliant Blue dye.

EXAMPLE 2 Expression by Via dß Gene Therapy The fibroblasts of a subject were obtained by skin biopsy. The resulting tissue was placed in the tissue culture medium and separated into small pieces. Small fragments of tissue were placed on a wet surface of a tissue culture flask, approximately ten pieces were placed in each flask. The flask was turned upside down, closed tightly and left at room temperature overnight. After 24 hour-s at room temperature, the flask was inverted and the tissue fragments remained fixed at the bottom of the flask and fresh medium (for example, Ham's F12 medium), with 10% FBS, penicillin and Streptomycin was added. This was then incubated at 37 ° C for about a week. At this time, the fresh medium was added and changed subsequently every several days. After two more weeks in culture, a monolayer of fibroblasts emerged. The monolayer was trypsinized and related in larger flasks. PMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988) flanked by the long terminal repeats of the murine sarcoma virus, from Moloney, was digested with EcoRI and HindlII and subsequently treated with phosphatase calf intestine The linear vector was fractionated on agarose gel and purified using glass beads The cDNA encoding a polypeptide of the present invention was amplified using the PCR primers corresponding to the 5 'and 3' end sequences. ', respectively. The 5 'primer containing an EcoRI site and the 3' primer containing a HindIII site. Equal amounts of the Moloney virus linear skeleton of murine sarcoma, and the EcoRI and HindIII fragment were added together, in the presence of the T4 DNA ligase. The resulting mixture was maintained under conditions appropriate for the ligand of the two fragments. The ligation mixture was used to transform HB101 bacteria, which were placed on agar containing kanamycin in order to confirm that the vector has the gene of interest inserted properly. The amphotropic amphotropic pA317 or GP + aml2 packaging cells were grown in tissue culture at the confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene was then added to the medium and the packaging cells were transduced with the vector. The packaging cells now produce infectious viral particles that contain the gene (the packaging cells are now named as the producer cells).

Fresh medium was added to the transduced producer cells, and then the medium was collected from a 10 cm plate of confluent producer cells. • The spent medium, which contains the infectious viral particles, was filtered through a millipore filter to remove the detached production cells and this means was then used to infect the fibroblast cells. The medium was removed from the sub-confluent plate of fibroblasts and rapidly replaced with the medium from the producer cells. This medium was removed and replaced with fresh medium. If the virus titer is high, then virtually all fibroblasts will be infected and a selection is not required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. The treated fibroblasts were then injected into the host, either alone or after they had grown to confluence in 3 microcarrier spheres of cytodex. The fibroblasts now produce the protein product.

Example 3 Bacterial Expression and Purification of VEGF3 The DNA of the deposited clone, which encodes the VEGF3 was initially amplified using oligonucleotide primers from the PCR, which corresponds to the 5 'sequences of the processed VEGF3 protein (minus the signal peptide sequence) and the 3' vector sequences to the VGEF3 gene. Additional nucleotides corresponding to VEGF3 were added to the 5 'and 3' sequences, respectively. The primer of the 5 'oligonucleotide has the 5' sequence GACTGCATGCACCAGA GGAAAGTGGTGTC (SEQ ID NO: 6) contains a restriction enzyme site followed by the sequence encoding VEGF3, starting from the presumed terminal amino acid of the processed protein codon. The 3 ', 5' sequence GACTAGATCTCCTTCGCAGCTTCCGGCAC 3 '(SEQ ID NO: 7) contains sequences complementary to the BglII site located 3' to the VEGF3 DNA insert. The restriction enzyme sites in the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA 91311). PQE-70 encodes antibiotic resistance (Ampr) a replication bacterial origin (ori), an IPTG regulator (P / O) promoter operator, a ribosome binding site (RBS), a 6-His tag, and restriction enzyme sites. The pQE-9 was then digested with Sphl and BglII. The amplified sequences were ligated into pQE-70, and inserted into the frame with the sequence encoding the histidine tag and the RBS. The ligation mixture was then used to transform E. coli strain M15 / rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. et al. Molecular Cloning: A Labora tory Manual, Cold, Spring Laboratory Press (1989). M15 / rep4 contains multiple copies of plasmid pREP4, which expresses the lacl repressor and also confers resistance to kanamycin (Kanr). Transformants were identified by their ability to grow on LB plates and colonies resistant to ampicillin / kanamycin were selected. Plasmid DNA was isolated and confirmed by restriction analysis. The clones containing the desired builders were grown overnight (0 / N) in a liquid culture in the LB medium supplemented with both Amp (100 ug / ml) and Kan (25 ug / ml). The O / N culture was used to inoculate a large culture at a ratio of 1: 100 to 1: 250. The cells grew at an optical density of 600 (D.O.600) between 0.4 and 0.6. The IPTG ("isopropyl-B-D-thiogalacto-pyranoside") was then added to a final concentration of 1 mM. The IPTG induces by the inactivation the lacl repressor, clarifying the P / O that guides the expression of the augmented gene. The cells grew for 3 to 4 extra hours. The cells were then harvested by centrifugation. The cell pellet was solubilized in a chaotropic agent of 5 molar guanidine-HCl. After clarification, the solubilized VEGF3 was purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow firm binding by the proteins containing the 6-His tag (Hochuli, E. et al), J. Chromatography 411: 177-184 (1984)). VEGF3 was eluted from the column "in 6 molar guanidine-HCl, pH 5.0 and for the purpose of renaturation it was adjusted to 3 molar guanidine-HCl with 100 mM sodium phosphate, 10 mmolar glutathione (reduced) and glutathione 2 mmolar (oxidized) After incubation in this solution for 12 hours, the protein was dialyzed to 10 molar sodium phosphate Numerous modifications and variants of the present invention are possible in light of the above teachings and therefore , they are within the scope of the appended claims, the invention can be practiced in a manner other than that particularly described.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: Olsen and collaborators. 5 (ii) TITLE OF THE INVENTION: Growth Factor 3, Endothelial, Vascular (iii) NUMBER OF SEQUENCES: (iv) ADDRESS OF CORRESPONDENCE: 0 (A) RECIPIENT: CARELLA, BYRNE, BAIN, GILFILLAN, CECCI, STEWART & OLSTEIN (B) STREET: 6 BECKER FARM ROAD (C) CITY; ROSELAND (D) STATE: NEW JERSEY 5 (E) COUNTRY: E.U.A. (F) POSTAL ZONE: 07068. (V) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: 3.5 INCH DISKET (B) COMPUTER: IBM PS / 2 0 (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE : WORD PERFECT 5.1 (Vi) CURRENT DATA OF THE APPLICATION (A) NUMBER OF THE APPLICATION: (B) DATE OF DEPOSIT: Concurrently 5 (C) CLASSIFICATION: (vii) INFORMATION OF THE EMPLOYEE / AGENT: (A) NAME: FERRARO, GREGORY D. (B) REGISTRATION NUMBER: 36,134 (C) REFERENCE NUMBER / EXPEDITION: 325800-0 (IX) TELECOMMUNICATIONS INFORMATION: (A) PHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION OF SEQ ID N0: 1 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 666 BASIC COUPLES (B) TYPE: NUCLEIC ACID 5 (C) CORD CLASS: SINGLE (D) TOPOLOGY: LINEAR (Ü) ) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: 0 ATGAGAAGGT GTAGAAXAAG TQGGAGGCCC CCGGCGCCCC CCGGTGTCCC CGCCCAGGCC 60 CCTGTCTCCC AGCCTGATGC CCCTGGCCAC CAGAGGAAAG TGGTGTCATG GATAGATGTG 120 TATACTCGCG CTACCTGCCA GCCCCGGGAG GTGGTGGTGC CCTTGACTGT GGACCTCATG 180 GGCACCGTGG CCAAACAGCr GGTGCCCAGC TGCGTGACTG TGCAGCGCTG TGGTGGCTGC 240 TGCCCTGACG ATGGCCTGGA GTGTGTGCCC ACTGGGCAGC ACCAAGTCCG GATGCAGATC 300 CTCATGATCC GGTACCCGAG CAGTCAGCTG GGGGAGATGT CCCTGGAAGA ACACAGCCAG 360 TGTGAATGCA GACCTAAAAA AAAGGACAGT GCTGTGAAGC CAGACAGGGC TGCTACTCCC 420 CACCACCGTC CCCAGCCCCG TtCTGTTCCG GGCTGGGACT CTGCCCCCGG AGCACCCTCC 480 5 CCAGCTGACA TCACCCAATC CCACTCCAGC CCCAGGCCCC TCTGCCCACG .CTGCACCCAG 540 CACCACCAGT GCCCTGACCC CCGGACCTGC CGCTGCCGCT GTCGACGCCG CAGCITCCTC 600 CGTTGTCAAG GGCGGGGCTT AGAGCTCAAC CCAGACACCT GCAGGTGCCG GAAGCTGCGA 660 AGGTGA 666 0 (2) INFORMATION OF SEQ ID NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 221 AMINO ACIDS (B) TYPE: AMINO ACIDS (C) LACE CLASS: 5 (D) TOPOLOGY: LINEAR (Ü) TYPE OF MOLECULE: PROTEIN (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Arg Arg Cys- Arg Lie Ser Gly Arg Pro Pro Ala Pro Pro Gly S? O 15 Val Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His 20. 25 30 Gln Arg Lys Val Val Ser Trp lie Asp Val Tyr Thr Arg Ala Thr 35 40 45 Cys Gln Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met 50 55 60 Gly Thr Val Wing Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln 65 70 75 Arg Cys Gly Gly Cys Cys Pro Asp Asp Gly Leu Glu Cy? Val Pro 80 85 90 Thr Gly Gln His Gln Val Arg Met Gln lie Leu Met He Arg Tyr 95 • 100 105 Pro Ser Ser Gln Leu Gly Glu Met Ser Leu Glu Glu His Ser Gln 110 115 120 Cye Glu Cys Arg Pro Lys Lys Lys Asp Ser Wing Val Lys Pro Asp 125 130 135 Arg Ala Ala Thr Pro His His Arg Pro Gln Pro Arg Ser Val Pro 140 145 150 Gly Trp Asp Be Wing Pro Gly Wing Pro Pro Pro Wing Aep He Thr 155 160 165 Gln Ser His Being Pro Pro Arg Pro Leu Cys Pro Arg Cys Thr Gln 170 175 180 His His Gln Cys Pro Asp Pro Arg Thr Cys Arg 'Cye Arg Cys Arg 185 190 195 Arg Arg "Being Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu Leu Asn 200 205 210 Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg 215 220

Claims (20)

1. CLAIMS 1. An isolated polynucleotide, comprising a member selected from the group consisting of: (a) a polynucleotide, which encodes the polypeptide which, as set forth in SEQ ID NO: 2; (b) a polynucleotide, capable of hybridizing to, and which is at least 70% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of this polynucleotide of (a) or (b).
2. 2. The polynucleotide of claim 1, wherein this polynucleotide is DNA.
3. 3. The polynucleotide of claim 2, which encodes the polypeptide as set forth in SEQ ID NO: 2
4. 4. The polynucleotide of claim 2, which encodes the polypeptide as set forth in SEQ ID NO: 2.
5. 5. The polynucleotide of claim 2, which encodes the polypeptide as set forth in SEQ ID NO: 2.
6. 6. An isolated polynucleotide, comprising a member selected from the group consisting of: (a) a polynucleotide, which encodes a mature polypeptide encoded by the DNA contained in the deposited clone; (b) a polynucleotide, which encodes the polypeptide expressed by the DNA contained in the deposited clone; (c) a polynucleotide, capable of hybridizing to, and which is at least 70% identical to the polynucleotide of (a) or (b); and (d) a polynucleotide fragment of the polynucleotides of (a), (b) or (c).
7. 7. A vector containing the DNA of claim 2.
8. 8. A host cell, genetically treated with the vector of claim 7.
9. 9. A method for producing a polypeptide, which comprises: expressing from the host cell of claim 8 the polypeptide encoded by this DNA.
10. 10. A method for producing cells capable of expressing a polypeptide, comprising transforming or transfecting the cells with the vector of claim 7.
11. 11. A polypeptide, comprising a member selected from the group consisting of (i) a polypeptide, having the deduced amino acid sequence of SEQ ID NO: 2 and its fragments, analogs and derivatives; e (ii) a polypeptide comprising amino acids 1 to 221 of SEQ ID NO: 2; and (iii) a polypeptide encoded by the cDNA of the deposited clone and the fragments, analogs and derivatives of this polypeptide.
12. 12. A compound, effective as an agonist of the polypeptide of claim 11.
13. 13. An effective compound as an antagonist against the polypeptide of claim 11.
14. 14. A method for the treatment of a patient in need of PGSG-1, this method comprises: administering to the patient a therapeutically effective amount of the polypeptide of claim 11.
15. 15. The method of claim 14, wherein the therapeutically effective amount of the polypeptide is administered by supplying the patient with the DNA encoding the polypeptide and expressing this polypeptide in vivo.
16. 16. A method for the treatment of a patient in need of VEGF3, this method comprises: administering to the patient a therapeutically effective amount of the compound of claim 12.
17. 17. A method for the treatment of a patient in need of inhibiting VEGF3, this method comprises: administering to the patient a therapeutically effective amount of the antagonist of claim 13.
18. 18. A method for diagnosing a disease or susceptibility to a disease related to the expression of the polypeptide of claim 11, this method comprises: determining a mutation in the nucleic acid sequence encoding the polypeptide.
19. 19. A diagnostic method, which comprises: analyzing the presence of the polypeptide of claim 11, in a sample derived from a host.
20. 20. A method for identifying compounds that bind to and activate or inhibit a receptor for the polypeptide of claim 11, this method comprises: contacting a cell expressing on its surface a polypeptide receptor, this receptor is associated with a second component, capable of supplying a detectable signal, in response to the binding of a compound to the receptor, with a compound to be classified, under conditions that allow binding to the receptor; and determining whether the compound binds to and activates or inhibits the receptor, by detecting the presence or absence of a signal generated from the interaction of the compound with the receptor.